A Universal Surrogate Reporter for Efficient Enrichment of CRISPR/Cas9-Mediated Homology-Directed Repair in Mammalian Cells
A Universal Surrogate Reporter for Efficient Enrichment of CRISPR/Cas9-Mediated Homology-Directed Repair in Mammalian Cells
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用于在哺乳动物细胞中有效富集 CRISPR/Cas9 介导的同源定向修复的通用替代报告基因
DOI：
10.1016/j.omtn.2019.12.021
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发表时间：
2019-12
期刊：
影响因子：
--
通讯作者：
Zhiying Zhang
中科院分区：
文献类型：
--
作者：
Nana Yan;Yongsen Sun;Yuanyuan Fang;Jingrong Deng;Lu Mu;Kun Xu;Joe S. Mymryk;Zhiying Zhang
CRISPR/Cas9介导的同源定向修复（HDR）可以用来精确地改造哺乳动物基因组。然而，HDR固有的低效率常常妨碍识别期望的修饰细胞。在这里，我们开发了一种新型的通用替代报告系统，其有效地富集由CRISPR/Cas9诱导的HDR事件产生的遗传修饰的细胞（即，“HDR-USR”系统）。这种基于附加型的报告基因可以通过HDR自切割和自修复，以产生功能性嘌呤霉素选择盒，而不损害基因组完整性。将HDR-USR系统共转染到宿主细胞中和瞬时嘌呤霉素选择有效地实现了HDR修饰的细胞的富集。我们在不同的人类细胞系中的16个位点和两个啮齿动物细胞系中的一个位点测试了该系统的精确点突变。该系统在单个基因座（高达20.7倍）和一次两个基因座（42%的编辑效率，而对照中为零）处表现出HDR效率的显著改善，以及在测试基因座处大大改善的敲入效率（8.9倍）和双等位基因缺失（35.9倍）。通过酵母Rad 52和线性单链/双链DNA供体的共表达实现了进一步的增加。总之，我们的HDR-USR系统提供了一种简单、稳健和有效的替代报告基因，用于在不同细胞系中的各种靶向基因座上富集CRISPR/Cas9诱导的基于HDR的精确基因组编辑。
CRISPR/Cas9-mediated homology-directed repair (HDR) can be leveraged to precisely engineer mammalian genomes. However, the inherently low efficiency of HDR often hampers to identify the desired modified cells. Here, we developed a novel universal surrogate reporter system that efficiently enriches for genetically modified cells arising from CRISPR/Cas9-induced HDR events (namely, the "HDR-USR" system). This episomally based reporter can be self-cleaved and self-repaired via HDR to create a functional puromycin selection cassette without compromising genome integrity. Co-transfection of the HDR-USR system into host cells and transient puromycin selection efficiently achieves enrichment of HDR-modified cells. We tested the system for precision point mutation at 16 loci in different human cell lines and one locus in two rodent cell lines. This system exhibited dramatic improvements in HDR efficiency at a single locus (up to 20.7-fold) and two loci at once (42% editing efficiency compared to zero in the control), as well as greatly improved knockin efficiency (8.9-fold) and biallelic deletion (35.9-fold) at test loci. Further increases were achieved by co-expression of yeast Rad52 and linear single-/double-stranded DNA donors. Taken together, our HDR-USR system provides a simple, robust and efficient surrogate reporter for the enrichment of CRISPR/Cas9-induced HDR-based precision genome editing across various targeting loci in different cell lines.
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