Xiong Kai, Zhou Yan, Blichfeld Kristian Aabo, Hyttel Poul, Bolund Lars, Freude Kristine Karla, Luo Yonglun
1 Department of Veterinary Clinical and Animal Sciences, University of Copenhagen , Frederiksberg, Denmark .
2 Danish Regenerative Engineering Alliance for Medicine, Department of Biomedicine, Aarhus University , Aarhus, Denmark .
Cell Reprogram. 2017 Jun;19(3):189-198. doi: 10.1089/cell.2017.0006.
Specific activation of endogenous genes can be achieved by programmable artificial transcription factors (ATFs). In this study, we compared two artificial, programmable, clustered regularly interspaced short palindromic repeats (CRISPR)-based, ubiquitous transcription factors: deficient CRISPR-associated protein 9 (dCas9)-VP64 (CRISPRa) alone, or a combination of dCas9-VP64 and MS2-P65-HSF1 [synergistic activation mediator (SAM) system] mediated activation of five pluripotency genes: KLF4 (K), LIN28 (L), MYC (M), OCT4 (O), and SOX2 (S) in human cells (HEK293T, HeLa, HepG2, and primary fibroblasts). Activation potential was monitored using a luciferase reporter system and we found that both CRISPRa and SAM can efficiently activate the proximal promoter of all five genes. We also observed that the guide RNA (gRNA) target sites and number of gRNAs have a major effect on gRNA-guided activation efficiency. Furthermore, increased activation efficiency (>3-folds) could be achieved by the SAM system compared to CRISPRa. In addition, we discovered that only the SAM system could efficiently activate LIN28, OCT4, and SOX2 expression (up to 100-folds compared to coexpression with a scrambled gRNA) in primary human fibroblasts. This SAM-mediated activation of LOS can be stably maintained for over 20 days in fibroblasts cultured in either fibroblasts or stem cell medium. However, when attempting to use the SAM-LOS activation as an approach for induced pluripotent stem cells-reprogramming, no embryonic stem-like colonies could be obtained from these SAM fibroblasts. In conclusion, our study showed that CRISPR/Cas9-based ATFs are potent to activate and maintain transcription of endogenous human pluripotent genes. However, future improvements of the system are still required to improve activation efficiency and cellular reprogramming using ATFs.
通过可编程人工转录因子（ATF）可实现内源性基因的特异性激活。在本研究中，我们比较了两种基于人工可编程成簇规律间隔短回文重复序列（CRISPR）的普遍存在的转录因子：单独的缺陷型CRISPR相关蛋白9（dCas9）-VP64（CRISPRa），或dCas9-VP64与MS2-P65-HSF1的组合[协同激活介质（SAM）系统]介导的人细胞（HEK293T、HeLa、HepG2和原代成纤维细胞）中五个多能性基因的激活：KLF4（K）、LIN28（L）、MYC（M）、OCT4（O）和SOX2（S）。使用荧光素酶报告系统监测激活潜力，我们发现CRISPRa和SAM均可有效激活所有五个基因的近端启动子。我们还观察到，引导RNA（gRNA）靶位点和gRNA数量对gRNA引导的激活效率有重大影响。此外，与CRISPRa相比，SAM系统可实现更高的激活效率（>3倍）。此外，我们发现只有SAM系统能够在原代人成纤维细胞中有效激活LIN28、OCT4和SOX2的表达（与用乱序gRNA共表达相比，激活倍数高达100倍）。这种SAM介导的LOS激活在成纤维细胞或干细胞培养基中培养的成纤维细胞中可稳定维持超过20天。然而，当尝试将SAM-LOS激活用作诱导多能干细胞重编程的方法时，从这些SAM成纤维细胞中无法获得胚胎干细胞样集落。总之，我们的研究表明，基于CRISPR/Cas9的ATF能够有效激活并维持内源性人类多能性基因的转录。然而，仍需要对该系统进行进一步改进，以提高使用ATF的激活效率和细胞重编程能力。
