Jordan Family Center for Blood and Marrow Transplantation and Cellular Therapies, Children's Hospital Oakland Research Institute, Oakland, CA 94609, USA ; Berkeley Stem Cell Center, University of California at Berkeley, Berkeley, CA 94720, USA.
Stem Cell Reports. 2013 Aug 15;1(3):218-25. doi: 10.1016/j.stemcr.2013.07.002. eCollection 2013.
MicroRNAs are important gene regulators involved in many biological processes, including stemness maintenance and cellular reprogramming. Current methods used in loss-of-function studies of microRNAs mainly include locked nucleic acid (LNA) oligonucleotides and miRZip inhibitors, which have several limitations. Due to their unique gene structures and small sizes, there is no efficient or simple strategy to knock down or knock out microRNAs or whole microRNA clusters. Here, we demonstrate knockdown of the miR-302/367 cluster by using the Kruppel-associated box repressor domain fused with specific transcription activator-like effectors (TALEs) designed to bind the miR-302/367 cluster promoter. We also designed two pairs of TALE nucleases (TALENs) to efficiently delete the miR-302/367 cluster in primary human fibroblasts and determined that knockout of the miR-302/367 cluster completely blocked induced pluripotent stem cell (iPSC) generation. Together, our results demonstrate that TALE-based transcriptional repressor and TALENs are two promising approaches for loss-of-function studies of microRNA clusters in somatic cells and pluripotent stem cells.
微小 RNA 是参与许多生物学过程的重要基因调控因子，包括干性维持和细胞重编程。目前用于微小 RNA 功能丧失研究的方法主要包括锁核酸（LNA）寡核苷酸和 miRZip 抑制剂，这些方法存在几个局限性。由于微小 RNA 独特的基因结构和较小的尺寸，没有有效的或简单的策略来敲低或敲除微小 RNA 或整个微小 RNA 簇。在这里，我们通过使用与特异性转录激活因子样效应物（TALEs）融合的 Kruppel 相关盒抑制剂来证明 miR-302/367 簇的敲低，这些 TALEs 被设计用于结合 miR-302/367 簇启动子。我们还设计了两对 TALE 核酸酶（TALENs），以有效地在原代人成纤维细胞中删除 miR-302/367 簇，并确定 miR-302/367 簇的敲除完全阻止了诱导多能干细胞（iPSC）的产生。总之，我们的结果表明，基于 TALE 的转录抑制剂和 TALENs 是研究体细胞和多能干细胞中微小 RNA 簇功能丧失的两种有前途的方法。
