Generation and Characterization of a MYF5 Reporter Human iPS Cell Line Using CRISPR/Cas9 Mediated Homologous Recombination.
Generation and Characterization of a MYF5 Reporter Human iPS Cell Line Using CRISPR/Cas9 Mediated Homologous Recombination.
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DOI：
10.1038/srep18759
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发表时间：
2016-01-05
影响因子：
4.6
通讯作者：
Darabi R
中科院分区：
文献类型：
--
作者：
Wu J;Hunt SD;Xue H;Liu Y;Darabi R
人类iPS细胞在疾病建模和退行性疾病（包括肌营养不良症）的治疗方面具有巨大的前景。虽然少数研究小组已经将其用于骨骼肌分化，但大多数都是基于基因过表达或长期间充质分化以及肌源性细胞的回顾性鉴定。因此，本研究旨在产生MYF 5的敲入报告人iPS细胞系，作为早期成肌特异性基因，以允许从人iPS细胞中前瞻性地鉴定和纯化成肌祖细胞。通过使用CRISPR/Cas9双切口酶策略，使用同源重组将2A-GFP报告基因插入MYF 5基因的终止密码子之前。这种方法允许在人类iPS细胞中高效地框内靶向MYF 5。此外，为了证明报告子功能，使用新的死Cas9-VP 160转录激活剂诱导内源性MYF 5表达。诱导的克隆表现出适当的MYF 5-GFP共表达。最后，为了确认分化潜力，通过胚状体方法分化报告人iPS克隆，并分选和表征MYF 5-GFP+肌细胞。这些数据为产生肌源性基因的敲入报告基因人iPS细胞系提供了有价值的指导方针，这些细胞系可用于疾病建模、药物筛选、基因校正和未来的体内应用。
Human iPS cells hold great promise for disease modeling and treatment of degenerative disorders including muscular dystrophies. Although a few research groups have used them for skeletal muscle differentiation, most were based on gene over-expression or long-term mesenchymal differentiation and retrospective identification of myogenic cells. Therefore, this study was aimed to generate a knock-in reporter human iPS cell line for MYF5, as an early myogenic specification gene, to allow prospective identification and purification of myogenic progenitors from human iPS cells. By using a CRISPR/Cas9 double nickase strategy, a 2A-GFP reporter was inserted before the stop codon of the MYF5 gene using homologous recombination. This approach allowed for highly efficient in-frame targeting of MYF5 in human iPS cells. Furthermore, in order to prove the reporter function, endogenous MYF5 expression was induced using a novel dead Cas9-VP160 transcriptional activator. Induced clones demonstrated appropriate MYF5-GFP co-expression. Finally, to confirm the differentiation potential, reporter human iPS clones were differentiated through embryoid body method and MYF5-GFP+ myogenic cells were sorted and characterized. These data provides valuable guidelines for generation of knock-in reporter human iPS cell lines for myogenic genes which can be used for disease modeling, drug screening, gene correction and future in vivo applications.