Laboratory of Experimental Immunology, BioMediTech Institute and Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland.
Laboratory of Anatomy, Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland.
PLoS One. 2018 Apr 23;13(4):e0196238. doi: 10.1371/journal.pone.0196238. eCollection 2018.
CRISPR-Cas9 technology is routinely applied for targeted mutagenesis in model organisms and cell lines. Recent studies indicate that the prokaryotic CRISPR-Cas9 system is affected by eukaryotic chromatin structures. Here, we show that the likelihood of successful mutagenesis correlates with transcript levels during early development in zebrafish (Danio rerio) embryos. In an experimental setting, we found that guide RNAs differ in their onset of mutagenesis activity in vivo. Furthermore, some guide RNAs with high in vitro activity possessed poor mutagenesis activity in vivo, suggesting the presence of factors that limit the mutagenesis in vivo. Using open access datasets generated from early developmental stages of the zebrafish, and guide RNAs selected from the CRISPRz database, we provide further evidence for an association between gene expression during early development and the success of CRISPR-Cas9 mutagenesis in zebrafish embryos. In order to further inspect the effect of chromatin on CRISPR-Cas9 mutagenesis, we analysed the relationship of selected chromatin features on CRISPR-Cas9 mutagenesis efficiency using publicly available data from zebrafish embryos. We found a correlation between chromatin openness and the efficiency of CRISPR-Cas9 mutagenesis. These results indicate that CRISPR-Cas9 mutagenesis is influenced by chromatin accessibility in zebrafish embryos.
CRISPR-Cas9 技术常用于对模式生物和细胞系进行靶向诱变。最近的研究表明，原核 CRISPR-Cas9 系统受到真核染色质结构的影响。在这里，我们表明在斑马鱼（Danio rerio）胚胎的早期发育过程中，成功诱变的可能性与转录水平相关。在实验环境中，我们发现向导 RNA 在体内诱变活性的起始时间存在差异。此外，一些具有高体外活性的向导 RNA 在体内具有较差的诱变活性，这表明存在限制体内诱变的因素。我们使用从斑马鱼早期发育阶段生成的公开访问数据集以及从 CRISPRz 数据库中选择的向导 RNA，进一步提供了证据表明，在早期发育过程中基因表达与 CRISPR-Cas9 对斑马鱼胚胎的诱变成功之间存在关联。为了进一步检查染色质对 CRISPR-Cas9 诱变的影响，我们使用来自斑马鱼胚胎的公开可用数据分析了所选染色质特征对 CRISPR-Cas9 诱变效率的影响。我们发现染色质开放性与 CRISPR-Cas9 诱变效率之间存在相关性。这些结果表明，CRISPR-Cas9 诱变受到斑马鱼胚胎中染色质可及性的影响。
