HDAC inhibitors improve CRISPR-mediated HDR editing efficiency in iPSCs
HDAC inhibitors improve CRISPR-mediated HDR editing efficiency in iPSCs
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HDAC 抑制剂可提高 iPSC 中 CRISPR 介导的 HDR 编辑效率。
DOI：
10.1007/s11427-020-1855-4
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发表时间：
2021
期刊：
影响因子：
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通讯作者：
Zhang Xiao-Bing
中科院分区：
文献类型：
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作者：
Zhang Jian-Ping;Yang Zhi-Xue;Zhang Feng;Fu Ya-Wen;Dai Xin-Yue;Wen Wei;Zhang Beldon;Choi Hannah;Chen Wanqiu;Brown Meredith;Baylink David;Zhang Lei;Qiu Hongyu;Wang Charles;Cheng Tao;Zhang Xiao-Bing
基因组编辑的人类诱导多能干细胞（iPSCs）在治疗应用方面具有很大的前景。然而，低编辑效率阻碍了CRISPR-Cas9技术在创建敲除和同源定向修复（HDR）编辑的iPSC系中的应用，特别是对于沉默基因。这部分是由于染色质压缩，不可避免地限制了Cas9进入目标DNA。在我们研究的六种HDAC抑制剂中，伏立诺他或亚甲基苯胺羟肟酸（SAHA）在开放和关闭位点的HDR效率最高，毒性可接受。由于HDAC抑制剂介导的Cas9和sgRNA表达的显著增加，HDAC抑制剂在打开和关闭位点上同样提高了非同源末端连接（NHEJ）编辑效率（约50%）。然而，我们观察到，相对于开放染色质，封闭位点的HDR效率有了更实质性的提高（2.8倍对1.7倍的变化）。这些研究为iPSCs中沉默基因的hdr编辑提供了一种新的策略。
Genome-edited human induced pluripotent stem cells (iPSCs) hold great promise for therapeutic applications. However, low editing efficiency has hampered the applications of CRISPR-Cas9 technology in creating knockout and homology-directed repair (HDR)-edited iPSC lines, particularly for silent genes. This is partially due to chromatin compaction, inevitably limiting Cas9 access to the target DNA. Among the six HDAC inhibitors we examined, vorinostat, or suberoylanilide hydroxamic acid (SAHA), led to the highest HDR efficiency at both open and closed loci, with acceptable toxicity. HDAC inhibitors equally increased non-homologous end joining (NHEJ) editing efficiencies (∼50%) at both open and closed loci, due to the considerable HDAC inhibitor-mediated increase in Cas9 and sgRNA expression. However, we observed more substantial HDR efficiency improvement at closed loci relative to open chromatin (2.8 vs. 1.7-fold change). These studies provide a new strategy for HDR-editing of silent genes in iPSCs.