Homozygous might be hemizygous: CRISPR/Cas9 editing in iPSCs results in detrimental on-target defects that escape standard quality controls.
Homozygous might be hemizygous: CRISPR/Cas9 editing in iPSCs results in detrimental on-target defects that escape standard quality controls.
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DOI：
10.1016/j.stemcr.2022.02.008
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发表时间：
2022-04-12
影响因子：
5.9
通讯作者：
Kiskinis, Evangelos
中科院分区：
文献类型：
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作者：
Simkin, Dina;Papakis, Vasileios;Bustos, Bernabe, I;Ambrosi, Christina M.;Ryan, Steven J.;Baru, Valeriya;Williams, Luis A.;Dempsey, Graham T.;McManus, Owen B.;Landers, John E.;Lubbe, Steven J.;George, Alfred L., Jr.;Kiskinis, Evangelos
使用CRISPR/Cas9精确编辑人类诱导多能干细胞（iPSC）系基因组的能力使得能够开发出能够解决基因型与表型关系的细胞模型。虽然基因组编辑正在成为基于iPSC的疾病建模研究中的一个重要工具，但编辑细胞的质量控制工作流程尚未建立。此外，最近在CRISPR/Cas9编辑的基因座中发现了通过DNA修复机制发生的大的靶向缺失和插入。然而，这些事件在人类iPSC中的频率仍然不清楚，因为它们很难检测到。我们检查了靶向9个基因座后产生的27个iPSC克隆，发现33%获得了大的靶基因组缺陷，包括插入和杂合性丢失。重要的是，所有缺陷都逃脱了标准PCR和桑格测序分析。我们描述了一种具有成本效益的质量控制策略，该策略成功地鉴定了所有具有有害靶事件的编辑克隆，并可以促进基于iPSC的研究的完整性。对在跨4个基因编辑9个基因组基因座后产生的27个iPSC克隆的检查CRISPR/Cas9编辑的iPSC系表现出大的中靶缺陷的高发生率大多数破坏性中靶插入是通过PCR/桑格测序检测不到的用于检测中靶缺陷的成本有效的质量控制测定的描述在本研究中，Simkin及其同事研究了CRISPR/Cas9介导的有害靶向效应在被认为是准确编辑的人类iPSC克隆中的发生。在33%的情况下，编辑的克隆获得了大的靶向结构变体，包括插入和微缺失。这些缺陷导致等位基因丢失，并使用标准PCR/桑格测序逃避检测。他们描述了准确筛选靶效应的方法。
The ability to precisely edit the genome of human induced pluripotent stem cell (iPSC) lines using CRISPR/Cas9 has enabled the development of cellular models that can address genotype to phenotype relationships. While genome editing is becoming an essential tool in iPSC-based disease modeling studies, there is no established quality control workflow for edited cells. Moreover, large on-target deletions and insertions that occur through DNA repair mechanisms have recently been uncovered in CRISPR/Cas9-edited loci. Yet the frequency of these events in human iPSCs remains unclear, as they can be difficult to detect. We examined 27 iPSC clones generated after targeting 9 loci and found that 33% had acquired large, on-target genomic defects, including insertions and loss of heterozygosity. Critically, all defects had escaped standard PCR and Sanger sequencing analysis. We describe a cost-efficient quality control strategy that successfully identified all edited clones with detrimental on-target events and could facilitate the integrity of iPSC-based studies. Examination of 27 iPSC clones generated after editing 9 genomic loci across 4 genes CRISPR/Cas9-edited iPSC lines exhibit high incidence of large on-target defects Majority of damaging on-target insertions are undetectable by PCR/Sanger sequencing Description of cost-efficient quality control assays to detect on-target defects In this study, Simkin and colleagues investigate the occurrence of CRISPR/Cas9-mediated deleterious on-target effects in human iPSC clones deemed to be accurately edited. In 33% of cases, edited clones had acquired large on-target structural variants, including insertions and microdeletions. These defects caused allelic dropout and escaped detection using standard PCR/Sanger sequencing. They describe methods to accurately screen for on-target effects.
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影响因子：
7
作者：
Cho SW;Kim S;Kim Y;Kweon J;Kim HS;Bae S;Kim JS
通讯作者：
Kim JS
影响因子：
4.5
作者：
Keller MC;Simonson MA;Ripke S;Neale BM;Gejman PV;Howrigan DP;Lee SH;Lencz T;Levinson DF;Sullivan PF;Schizophrenia Psychiatric Genome-Wide Association Study Consortium
通讯作者：
Schizophrenia Psychiatric Genome-Wide Association Study Consortium
影响因子：
56.9
作者：
Bibikova, M;Beumer, K;Carroll, D
通讯作者：
Carroll, D
影响因子：
48
作者：
Ikeda, Kazuya;Uchida, Nobuko;Porteus, Matthew H.
通讯作者：
Porteus, Matthew H.
DOI：
10.3791/53583
发表时间：
2016-02-02
期刊：
Journal of visualized experiments : JoVE
影响因子：
--
作者：
Blair JD;Bateup HS;Hockemeyer DF
通讯作者：
Hockemeyer DF