Enhanced CRISPR/Cas9-mediated precise genome editing by improved design and delivery of gRNA, Cas9 nuclease, and donor DNA
Enhanced CRISPR/Cas9-mediated precise genome editing by improved design and delivery of gRNA, Cas9 nuclease, and donor DNA
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DOI：
10.1016/j.jbiotec.2016.11.011
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发表时间：
2017-01-10
影响因子：
4.1
通讯作者：
Chesnut, Jonathan D.
中科院分区：
文献类型：
--
作者：
Liang, Xiquan;Potter, Jason;Chesnut, Jonathan D.
虽然在哺乳动物细胞中基于CRISPR的基因敲除已被证明是非常有效的，但通过细胞同源性定向修复（HDR）途径精确插入遗传元件仍然是无缝基因组编辑的限速步骤。在本文所述的条件下，我们在HEK 293细胞中实现了高达56%的靶向整合效率，其中插入了多达6个核苷酸。在诱导多能干细胞（iPSC）中，我们通过共同递送Cas9 RNP和供体DNA实现了高达45%的精确基因组编辑率。此外，利用3'端突出的短双链DNA寡核苷酸可以使较长的FLAG表位标签沿着与限制性位点的整合率高达50%，我们提出了一个模型，该模型有利于供体DNA的设计，其变化尽可能接近切割位点。对于小的变化如SNP或短插入，具有30个碱基同源臂的不对称单链供体分子在插入/修复盒的3'端和在5'端具有大于40个碱基的同源性似乎是有利的。对于较大的插入，如表位标签，具有30个碱基的突出3'同源臂的dsDNA供体是有利的。在这两种情况下，用硫代磷酸酯修饰保护供体DNA的末端提高了编辑效率。(C)2016年6月，作者。由Elsevier B. V.出版。这是一篇开放获取的文章，在CC BY许可证下
While CRISPR-based gene knock out in mammalian cells has proven to be very efficient, precise insertion of genetic elements via the cellular homology directed repair (HDR) pathway remains a rate-limiting step to seamless genome editing. Under the conditions described here, we achieved up to 56% targeted integration efficiency with up to a six-nucleotide insertion in HEK293 cells. In induced pluripotent stem cells (iPSCs), we achieved precise genome editing rates of up to 45% by co-delivering the Cas9 RNP and donor DNA. In addition, the use of a short double stranded DNA oligonucleotide with 3' overhangs allowed integration of a longer FLAG epitope tag along with a restriction site at rates of up to 50%.We propose a model that favors the design of donor DNAs with the change as close to the cleavage site as possible. For small changes such as SNPs or short insertions, asymmetric single stranded donor molecules with 30 base homology arms 3' to the insertion/repair cassette and greater than 40 bases of homology on the 5' end seems to be favored. For larger insertions such as an epitope tag, a dsDNA donor with protruding 3' homology arms of 30 bases is favored. In both cases, protecting the ends of the donor DNA with phosphorothioate modifications improves the editing efficiency. (C) 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license