Enhancing site-specific DNA integration by a Cas9 nuclease fused with a DNA donor-binding domain.
Enhancing site-specific DNA integration by a Cas9 nuclease fused with a DNA donor-binding domain.
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通过与 DNA 供体结合域融合的 Cas9 核酸酶增强位点特异性 DNA 整合
DOI：
10.1093/nar/gkaa779
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发表时间：
2020-10-09
影响因子：
14.9
通讯作者：
Rong Z
中科院分区：
文献类型：
--
作者：
Ma S;Wang X;Hu Y;Lv J;Liu C;Liao K;Guo X;Wang D;Lin Y;Rong Z
摘要CRISPR/CAS系统广泛用于基因组编辑。然而，稳健和有针对性地插入DNA片段仍然是一个挑战。在这里，我们提出了一个融合核酸酶(Cas9-N57)，通过睡美人转座酶的融合DNA结合域将DNA片段拴在Cas9/sgRNA复合体上，从而增强位点特异性的DNA整合。插入是单向的和特异的，成功整合了长达12kb的DNA片段。作为对该系统的测试，Cas9-N57介导CD19特异性嵌合抗原受体(CD19-CAR)盒插入人T细胞的AAVS1位点，并通过同时介导致癌基因KrasG12D插入rosa26位点并干扰TrP53和Pten而诱发小鼠肝内胆管癌细胞。此外，基于AsCpf1(AsCas12a)和CjCas9的核酸酶-N57融合蛋白具有相似的活性。这些发现表明，CRISPR相关核酸酶-N57蛋白融合是一种强大的靶向DNA插入工具，在基因治疗应用中具有巨大的潜力。
Abstract The CRISPR/Cas system is widely used for genome editing. However, robust and targeted insertion of a DNA segment remains a challenge. Here, we present a fusion nuclease (Cas9-N57) to enhance site-specific DNA integration via a fused DNA binding domain of Sleeping Beauty transposase to tether the DNA segment to the Cas9/sgRNA complex. The insertion was unidirectional and specific, and DNA fragments up to 12 kb in length were successfully integrated. As a test of the system, Cas9-N57 mediated the insertion of a CD19-specific chimeric antigen receptor (CD19-CAR) cassette into the AAVS1 locus in human T cells, and induced intrahepatic cholangiocarcinoma in mice by simultaneously mediating the insertion of oncogenic KrasG12D into the Rosa26 locus and disrupting Trp53 and Pten. Moreover, the nuclease-N57 fusion proteins based on AsCpf1 (AsCas12a) and CjCas9 exhibited similar activity. These findings demonstrate that CRISPR-associated nuclease-N57 protein fusion is a powerful tool for targeted DNA insertion and holds great potential for gene therapy applications.
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一个 iCRISPR 平台，用于在人类多能干细胞中进行快速、可多重、可诱导的基因组编辑。
影响因子：
23.9
作者：
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影响因子：
16.6
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通讯作者：
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影响因子：
14.9
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通讯作者：
Flicek P
影响因子：
16.6
作者：
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通讯作者：
Saha K
影响因子：
64.8
作者：
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通讯作者：
Sadelain M