Using local chromatin structure to improve CRISPR/Cas9 efficiency in zebrafish.
Using local chromatin structure to improve CRISPR/Cas9 efficiency in zebrafish.
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利用局部染色质结构提高斑马鱼的 CRISPR/Cas9 效率
DOI：
10.1371/journal.pone.0182528
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Zhang Y
中科院分区：
文献类型：
--
作者：
Chen Y;Zeng S;Hu R;Wang X;Huang W;Liu J;Wang L;Liu G;Cao Y;Zhang Y
尽管CRISPR/Cas9已成功应用于斑马鱼，但对于不同的gRNA，已观察到相当大的效率差异。斑马鱼突变体筛选的工作量和成本在很大程度上取决于注射胚胎的突变率;因此，选择更有效的gRNA对于斑马鱼突变体构建尤为重要。除了序列特征之外，局部染色质结构可能对CRISPR/Cas9效率有影响，这在很大程度上尚未探索。在斑马鱼的唯一相关研究中，没有发现核小体组织对CRISPR/Cas9效率有影响，这与最近在体外和哺乳动物细胞系中的研究不一致。为了了解斑马鱼中局部染色质结构对CRISPR/Cas9效率的影响，我们首先确定CRISPR/Cas9主要在圆顶阶段之前引入基因组编辑。基于这一观察结果，我们重新分析了我们发表的核小体组织概况，并使用ATAC-seq技术在256细胞和圆顶阶段生成了染色质可及性概况。我们的研究表明，染色质可及性与CRISPR/Cas9效率呈正相关，但我们没有观察到核小体组织与CRISPR/Cas9效率之间的明确相关性。我们构建了一个基于局部染色质结构特征的斑马鱼gRNA选择在线数据库，该数据库可以证明有利于通过CRISPR/Cas9构建斑马鱼纯合突变体。
Although the CRISPR/Cas9 has been successfully applied in zebrafish, considerable variations in efficiency have been observed for different gRNAs. The workload and cost of zebrafish mutant screening is largely dependent on the mutation rate of injected embryos; therefore, selecting more effective gRNAs is especially important for zebrafish mutant construction. Besides the sequence features, local chromatin structures may have effects on CRISPR/Cas9 efficiency, which remain largely unexplored. In the only related study in zebrafish, nucleosome organization was not found to have an effect on CRISPR/Cas9 efficiency, which is inconsistent with recent studies in vitro and in mammalian cell lines. To understand the effects of local chromatin structure on CRISPR/Cas9 efficiency in zebrafish, we first determined that CRISPR/Cas9 introduced genome editing mainly before the dome stage. Based on this observation, we reanalyzed our published nucleosome organization profiles and generated chromatin accessibility profiles in the 256-cell and dome stages using ATAC-seq technology. Our study demonstrated that chromatin accessibility showed positive correlation with CRISPR/Cas9 efficiency, but we did not observe a clear correlation between nucleosome organization and CRISPR/Cas9 efficiency. We constructed an online database for zebrafish gRNA selection based on local chromatin structure features that could prove beneficial to zebrafish homozygous mutant construction via CRISPR/Cas9.
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DOI：
10.1242/dev.115584
发表时间：
2014-12
期刊：
Development (Cambridge, England)
影响因子：
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作者：
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