Histone deacetylase inhibitors (HDACis) affect DNA repair pathways by modulating multiple cellular machineries, including chromatin state, DNA repair factor modification, and the cell cycle. These machineries can differentially affect DNA repair outcomes. With the aim to investigate the impacts of HDACis on DNA repair following CRISPR/Cas9 cleavage from the mixed actions, we used two pan-HDACis, trichostatin A (TSA) and PCI-24781, to treat animal immortalized and primary cells, and studied CRISPR/Cas9-mediated genome editing results by nonhomologous end joining (NHEJ) and homology-directed repair (HDR) pathways. We first found that TSA and PCI-24781 increased NHEJ efficiency. However, further analysis of the total NHEJ events demonstrated that alternative end joining (alt-EJ) mainly contributed to the enhanced total NHEJ by HDACis. We then analyzed HDR efficiency with HDACi treatment and found that multiple HDR pathways, including homologous recombination, single strand annealing and single-stranded oligonucleotide (ssODN)-mediated HDR, were all increased with HDACi treatment. TSA also increased CRISPR-induced ssODN-mediated HDR rate in pig parthenogenetic embryos. Analyzing acetylation status of DNA repair factors showed that acetylation levels of classical NHEJ (c-NHEJ) factors KU70 and KU80 and alt-EJ factor PARP1 were significantly enhanced, but alt-EJ factor LIG3 and HDR factors Rad51 and Rad52 were not affected greatly, implying a differential impact on these repair pathways by HDACis. In addition, TSA and PCI-24781 can enrich cells in G2/M phase of the cell cycle which is beneficial for occurrence of HDR. These findings show that HDACis can effectively promote CRISPR-mediated homology-involved DNA repair, including HDR and alt-EJ pathways, through concerted action of multiple cellular machineries.

组蛋白脱乙酰基酶抑制剂（HDACis）通过调节多种细胞机制（包括染色质状态，DNA修复因子修饰和细胞周期）来影响DNA修复途径。这些机制可以差异地影响DNA修复结果。为了研究CRISPR / Cas9混合作用切割后HDACis对DNA修复的影响，我们使用了两种泛HDACis，曲古抑菌素A（TSA）和PCI-24781处理动物永生和原代细胞，并研究了CRISPR / Cas9介导的基因组编辑结果通过非同源末端连接（NHEJ）和同源性定向修复（HDR）途径完成。我们首先发现TSA和PCI-24781提高了NHEJ效率。但是，对所有NHEJ事件的进一步分析表明，替代末端连接（alt-EJ）主要是由HDACis增强了总NHEJ的作用。然后，我们分析了HDACi处理的HDR效率，发现HDACi处理增加了多个HDR途径，包括同源重组，单链退火和单链寡核苷酸（ssODN）介导的HDR。TSA还提高了猪孤雌生殖胚胎中CRISPR诱导的ssODN介导的HDR率。分析DNA修复因子的乙酰化状态表明，经典NHEJ（c-NHEJ）因子KU70和KU80和alt-EJ因子PARP1的乙酰化水平显着提高，但alt-EJ因子LIG3和HDR因子Rad51和Rad52受到的影响不大，暗示HDACis对这些修复途径有不同的影响。另外，TSA和PCI-24781可以使细胞周期的G2 / M期的细胞富集，这有利于HDR的发生。