Application of TALE-Based Approach for Dissecting Functional MicroRNA-302/367 in Cellular Reprogramming

  • Zhonghui Zhang
  • Wen-Shu Wu
Part of the Methods in Molecular Biology book series (MIMB, volume 1733)


MicroRNAs are small 18–24 nt single-stranded noncoding RNA molecules involved in many biological processes, including stemness maintenance and cellular reprogramming. Current methods used in loss-of-function studies of microRNAs have several limitations. Here, we describe a new approach for dissecting miR-302/367 functions by transcription activator-like effectors (TALEs), which are natural effector proteins secreted by Xanthomonas and Ralstonia bacteria. Knockdown of the miR-302/367 cluster uses the Kruppel-associated box repressor domain fused with specific TALEs designed to bind the miR-302/367 cluster promoter. Knockout of the miR-302/367 cluster uses two pairs of TALE nucleases (TALENs) to delete the miR-302/367 cluster in human primary cells. Together, both TALE-based transcriptional repressor and TALENs are two promising approaches for loss-of-function studies of microRNA cluster in human primary cells.

Key words

TALE TALEN Transcriptional repressor MicroRNA Cellular reprogramming Human cells 


  1. 1.
    Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116(2):281–297CrossRefPubMedGoogle Scholar
  2. 2.
    Johnston RJ, Hobert O (2003) A microRNA controlling left/right neuronal asymmetry in Caenorhabditis elegans. Nature 426(6968):845–849CrossRefPubMedGoogle Scholar
  3. 3.
    Liao B et al (2011) MicroRNA cluster 302–367 enhances somatic cell reprogramming by accelerating a mesenchymal-to-epithelial transition. J Biol Chem 286(19):17359–17364CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Robertson B et al (2010) Specificity and functionality of microRNA inhibitors. Silence 1(1):10CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Xia H, Ooi LL, Hui KM (2012) MiR-214 targets beta-catenin pathway to suppress invasion, stem-like traits and recurrence of human hepatocellular carcinoma. PLoS One 7(9):e44206CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Moscou MJ, Bogdanove AJ (2009) A simple cipher governs DNA recognition by TAL effectors. Science 326(5959):1501CrossRefPubMedGoogle Scholar
  7. 7.
    Hockemeyer D et al (2011) Genetic engineering of human pluripotent cells using TALE nucleases. Nat Biotechnol 29(8):731–734CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Zhang F et al (2011) Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nat Biotechnol 29(2):149–153CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Sanjana NE et al (2012) A transcription activator-like effector toolbox for genome engineering. Nat Protoc 7(1):171–192CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  1. 1.School of Life SciencesShanghai UniversityShanghaiChina
  2. 2.Division of Hematology/Oncology, Department of Medicine and Cancer CenterUniversity of Illinois at ChicagoChicagoUSA

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