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|Title:||Transcriptional activation of LGR5 gene by an engineered CRISPR-Cas9-based system induces hepatic-specific factors|
|Keywords:||Dead Cas9;Direct reprogramming;Yamanaka factors;Endogenous trans-activation;Gene regulation|
|Abstract:||Several new approaches for reprogramming or direct reprogramming somatic cells have been implemented during the last few years. Endogenous gene activation or repression can be achieved using dead clustered regularly interspaced short palindromic repeats associated protein 9 (dCas9) system fused with a transcriptional activating or repression domain. This study was undertaken to screen and validate efficient sgRNAs targeting reprogramming or direct reprogramming transcription factors by CRISPR-Cas9 based system. In this study, we designed and validated several individual single-guide RNA (sgRNA) targeting LGR5 and Yamanaka factors, such as Oct3/4, Sox2, c-Myc and Klf4, for effective transcriptional activation in mouse cells. Furthermore, we investigated the combination of effective sgRNAs for the synchronized effect of transcriptional activation on LGR5 gene and Yamanaka factors and achieved approximately 8.4-fold and 38-fold higher levels of mouse LGR5 and Oct3/4 upregulation, respectively, compared with the control. Further, we demonstrate that the activation LGR5 gene promoter induces known defined factors responsible for direct conversion of somatic cells to hepatocyte-like cells. We envision that our validation of effective sgRNAs will facilitate the development of mouse induced pluripotent stem cells (iPSCs) and novel findings of LGR5 as a potential candidate for direct conversion of somatic cells to hepatocyte-like cells.|
|ISSN:||0975-0967 (Online); 0972-5849 (Print)|
|Appears in Collections:||IJBT Vol.17(4) [October 2018]|
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