Effective control of large deletions after double-strand breaks by homology-directed repair and dsODN insertion.
Effective control of large deletions after double-strand breaks by homology-directed repair and dsODN insertion.
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通过同源定向修复和 dsODN 插入有效控制双链断裂后的大缺失。
DOI：
10.1186/s13059-021-02462-4
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发表时间：
2021-08-20
期刊：
影响因子：
12.3
通讯作者：
Zhang XB
中科院分区：
文献类型：
--
作者：
Wen W;Quan ZJ;Li SA;Yang ZX;Fu YW;Zhang F;Li GH;Zhao M;Yin MD;Xu J;Zhang JP;Cheng T;Zhang XB
在修复由CRISPR-Cas9切割引起的双链断裂（DSB）后，基因组损伤（例如大缺失）可能具有致病性后果。我们发现，大的缺失是普遍存在的，但依赖于编辑位点和细胞类型。人类原代T细胞显示出比造血干细胞和祖细胞（HSPC）更显著的缺失，而我们观察到诱导多能干细胞（iPSC）中的低水平。我们发现，同源性定向修复（HDR）与单链寡脱氧核苷酸（ssODNs）携带短同源性减少了几乎一半的缺失损伤，而腺相关病毒（AAV）的捐助者与长同源性减少了约80%的大缺失。在不存在HDR的情况下，通过NHEJ插入短双链ODN使缺失指数降低约60%。通过HDR和NHEJ及时桥接断裂末端大大降低了dsDNA切割的意外后果。这些策略可以在基因编辑应用中加以利用，以减少意想不到的结果。在线版本包含补充材料，可通过10.1186/s13059-021-02462-4获得。
After repairing double-strand breaks (DSBs) caused by CRISPR-Cas9 cleavage, genomic damage, such as large deletions, may have pathogenic consequences. We show that large deletions are ubiquitous but are dependent on editing sites and cell types. Human primary T cells display more significant deletions than hematopoietic stem and progenitor cells (HSPCs), whereas we observe low levels in induced pluripotent stem cells (iPSCs). We find that the homology-directed repair (HDR) with single-stranded oligodeoxynucleotides (ssODNs) carrying short homology reduces the deletion damage by almost half, while adeno-associated virus (AAV) donors with long homology reduce large deletions by approximately 80%. In the absence of HDR, the insertion of a short double-stranded ODN by NHEJ reduces deletion indexes by about 60%. Timely bridging of broken ends by HDR and NHEJ vastly decreases the unintended consequences of dsDNA cleavage. These strategies can be harnessed in gene editing applications to attenuate unintended outcomes. The online version contains supplementary material available at 10.1186/s13059-021-02462-4.
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