基因组编辑
生物
诱导多能干细胞
Cas9
清脆的
同源重组
同源定向修复
遗传学
计算生物学
基因组工程
基因座(遗传学)
基因
胚胎干细胞
DNA修复
核苷酸切除修复
作者
Renata Martin,Kazuya Ikeda,M. Kyle Cromer,Naoshige Uchida,Toshinobu Nishimura,Rosa Romano,Andrew J. Tong,Viktor T. Lemgart,Joab Camarena,Mara Pavel-Dinu,Camille Sindhu,Volker Wiebking,Sriram Vaidyanathan,Daniel P. Dever,Rasmus O. Bak,Anders Laustsen,Benjamin J. Lesch,Martin R. Jakobsen,Vittorio Sebastiano,Hiromitsu Nakauchi,Matthew H. Porteus
出处
期刊:Cell Stem Cell
[Elsevier]
日期:2019-05-01
卷期号:24 (5): 821-828.e5
被引量:123
标识
DOI:10.1016/j.stem.2019.04.001
摘要
Genome editing of human pluripotent stem cells (hPSCs) provides powerful opportunities for in vitro disease modeling, drug discovery, and personalized stem cell-based therapeutics. Currently, only small edits can be engineered with high frequency, while larger modifications suffer from low efficiency and a resultant need for selection markers. Here, we describe marker-free genome editing in hPSCs using Cas9 ribonucleoproteins (RNPs) in combination with AAV6-mediated DNA repair template delivery. We report highly efficient and bi-allelic integration frequencies across multiple loci and hPSC lines, achieving mono-allelic editing frequencies of up to 94% at the HBB locus. Using this method, we show robust bi-allelic correction of homozygous sickle cell mutations in a patient-derived induced PSC (iPSC) line. Thus, this strategy shows significant utility for generating hPSCs with large gene integrations and/or single-nucleotide changes at high frequency and without the need for introducing selection genes, enhancing the applicability of hPSC editing for research and translational uses.
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