Efficient CRISPR-Cas9-mediated generation of knockin human pluripotent stem cells lacking undesired mutations at the targeted locus.
Efficient CRISPR-Cas9-mediated generation of knockin human pluripotent stem cells lacking undesired mutations at the targeted locus.
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DOI：
10.1016/j.celrep.2015.04.007
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发表时间：
2015-05-12
期刊：
影响因子：
8.8
通讯作者：
Eggan K
中科院分区：
文献类型：
--
作者：
Merkle FT;Neuhausser WM;Santos D;Valen E;Gagnon JA;Maas K;Sandoe J;Schier AF;Eggan K
CRISPR-Cas9系统有可能彻底改变人类多能干细胞（hPSC）的基因组编辑，但其优势和缺陷仍然知之甚少。我们系统地测试了CRISPR-Cas9在hPSC中的16个不同基因组位点介导报告基因敲入的能力。我们观察到有效的基因靶向，但发现靶向克隆在靶向基因的两个等位基因处携带意外高频率的插入和缺失（indel）突变。这些插入缺失由Cas9核酸酶以及Cas9-D10 A单切口酶或双切口酶诱导，并且通常破坏基因功能。为了克服这个问题，我们设计了物理破坏或分离靶向等位基因处的CRISPR靶位点的策略，并开发了一种生物信息学管道来鉴定和消除在另一个等位基因处含有有害indel的克隆。这种双管齐下的方法使得能够可靠地产生在靶向基因座处不含不需要的突变的敲入hPSC报告细胞系。用CRISPR-Cas9表达质粒和靶向载体转染的人多能干细胞产生细胞群体，其中少数稳定整合赋予抗生素抗性的报告基因/选择盒。抗药性细胞在随机基因组位置（浅绿色）或在所需基因座（深绿色）稳定整合了报告基因/选择盒。PCR筛选可用于快速鉴定正确靶向的细胞，所述细胞可以在靶向和非靶向等位基因处都是完整的（黑色边界），或者可以具有“中靶”插入缺失（红色边界）。在两个等位基因处的深度测序可以鉴定正确靶向且不含不需要的插入缺失的所需克隆的子集。在测试的条件下，我们使用Cas9 dn介导靶向并且当我们设计通过插入报告基因/选择盒物理分离或破坏的CRISPR靶位点时，可靠地获得了完整且正确靶向的克隆。
The CRISPR-Cas9 system has the potential to revolutionize genome editing in human pluripotent stem cells (hPSCs), but its advantages and pitfalls are still poorly understood. We systematically tested the ability of CRISPR-Cas9 to mediate reporter gene knock-in at 16 distinct genomic sites in hPSCs. We observed efficient gene targeting but found that targeted clones carried an unexpectedly high frequency of insertion and deletion (indel) mutations at both alleles of the targeted gene. These indels were induced by Cas9 nuclease, as well as Cas9-D10A single or dual nickases, and often disrupted gene function. To overcome this problem, we designed strategies to physically destroy or separate CRISPR target sites at the targeted allele, and developed a bioinformatic pipeline to identify and eliminate clones harboring deleterious indels at the other allele. This two-pronged approach enables the reliable generation of knock-in hPSC reporter cell lines free of unwanted mutations at the targeted locus. Human pluripotent stem cells transfected with a CRISPR-Cas9 expression plasmid and targeting vectors give rise to a population of cells, of which a small minority stably integrate the reporter/selection cassette that confers antibiotic resistance. Drug resistant cells have stably integrated the reporter/selection cassette at a random genomic location (light green) or at the desired locus (dark green). PCR screening can be used to rapidly identify correctly targeted cells, which can either be intact at both the targeted and non-targeted allele (black border), or may habor “on-target” indels (red border). Deep sequencing at both alleles can identify the subset of desired clones that are both correctly targeted and free of unwanted indels. Under the conditions tested, we reliably obtained intact and correctly targeted clones using Cas9dn to mediate targeting and when we designed CRISPR target sites to be physically separated or destroyed by insertion of the reporter/selection cassette.
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