Genome-Scale CRISPRa Screen Identifies Novel Factors for Cellular Reprogramming
Genome-Scale CRISPRa Screen Identifies Novel Factors for Cellular Reprogramming
复制标题
DOI：
10.1016/j.stemcr.2019.02.010
复制
发表时间：
2019-03
影响因子：
5.9
通讯作者：
Jian Yang;S. S. Rajan-S.;Mathias J Friedrich;Guocheng Lan;X. Zou;H. Ponstingl;Dimitrios A. Garyfallos;Pentao Liu;A. Bradley;E. Metzakopian
中科院分区：
文献类型：
--
作者：
Jian Yang;S. S. Rajan-S.;Mathias J Friedrich;Guocheng Lan;X. Zou;H. Ponstingl;Dimitrios A. Garyfallos;Pentao Liu;A. Bradley;E. Metzakopian
致敏的上胚层干细胞（EpiSC）可以通过表达单个重编程因子而恢复为多能胚胎干细胞（ESC）样状态。我们使用CRISPR激活在EpiSC中进行基因组规模的重编程筛选，并确定了142个候选基因。我们的筛选总共验证了50个基因，以前不知道这些基因有助于重编程，我们选择其中的Sall1进行进一步研究。我们发现Sall 1增强了小鼠EpiSC和胚胎成纤维细胞的重编程，并且这些诱导的多能干细胞确实是完全多能的，包括嵌合小鼠的形成。我们还证明了Sall1与Nanogin重编程协同作用，并且ESCs中的过表达延迟了它们向EpiSCs的转化。最后，利用RNA测序，我们鉴定并验证Klf5和Fam189a2作为Sall1和Nanog的新下游靶点。总之，我们的工作证明了使用CRISPR技术理解介导复杂细胞过程（如重编程）的分子机制的力量。
Primed epiblast stem cells (EpiSCs) can be reverted to a pluripotent embryonic stem cell (ESC)-like state by expression of single reprogramming factor. We used CRISPR activation to perform a genome-scale, reprogramming screen in EpiSCs and identified 142 candidate genes. Our screen validated a total of 50 genes, previously not known to contribute to reprogramming, of which we choseSall1for further investigation. We show thatSall1augments reprogramming of mouse EpiSCs and embryonic fibroblasts and that these induced pluripotent stem cells are indeed fully pluripotent including formation of chimeric mice. We also demonstrate thatSall1synergizes withNanogin reprogramming and that overexpression in ESCs delays their conversion back to EpiSCs. Lastly, using RNA sequencing, we identify and validateKlf5andFam189a2as new downstream targets ofSall1andNanog. In summary, our work demonstrates the power of using CRISPR technology in understanding molecular mechanisms that mediate complex cellular processes such as reprogramming.