Genes & Genomics

, Volume 41, Issue 4, pp 491–498 | Cite as

Enhancement of gene knockdown efficiency by CNNC motifs in the intronic shRNA precursor

  • Seong Kyun Park
  • Yun Kee
  • Byung Joon HwangEmail author
Research Article



Short hairpin RNAs (shRNAs) expressed from vectors have been used as an effective means of exploiting the RNA interference (RNAi) pathway in mammalian cells. Of several methods to express shRNA, a method of transcribing shRNAs embedded in microRNA precursors has been more widely used than the one that directly expresses shRNA from RNA polymerase III promoters because the microRNA precursor form of shRNA is known to cause lower levels of cytotoxicity and off-target effects than the overexpressed shRNAs from the RNA polymerase III promoters.


We study the primary sequence features of microRNA precursors, which enhance their processing into mature form, helps design more potent shRNA precursors embedded in microRNA precursors.


We measure the enhancement of gene knockdown efficiency by adding CNNC motifs in the 3′ flanking region of shRNA precursor embedded in the human miR-30a microRNA precursor.


By systemically adding three CNNC motifs in the 3′ flanking region of shRNA precursor, we found that addition of two CNNC motifs saturates their enhanced knockdown ability of shRNA and that the CNNC motif in the + 17 to + 20 from the drosha cleavage site is most important for the shRNA-mediated target gene knock down. We also did see little knockdown of target gene expression by the shRNA precursor lacking CNNC motif.


Since genetic studies generally require techniques that could reduce gene expression at different degrees, the findings in this study will allow us to use RNAi for genetic studies of reducing gene expression at different degrees.


RNAi microRNA shRNA CNNC motif 



This work was supported by the SGER Program through the NRF by the Ministry of Education to Y. Kee (Grant Number NRF-2015R1D1A1A02060346); Basic Science Research Program through the NRF by the Ministry of Education (Grant Number NRF-2015R1D1A3A01015641), the grant from the National R&D Program for Cancer Control, Ministry of Health & Welfare, Republic of Korea (Grant Number HA17C0035), and 2015 Research Grant from Kangwon National University to B.J. Hwang.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.


  1. Abbas-Terki T, Blanco-Bose W, Déglon N et al (2002) Lentiviral-mediated RNA interference. Hum Gene Ther 13:2197–2201CrossRefGoogle Scholar
  2. Agrawal N, Dasaradhi PVN, Mohmmed A et al (2003) RNA interference: biology, mechanism, and applications. Microbiol Mol Biol Rev 67:657–685CrossRefGoogle Scholar
  3. Auyeung VC, Ulitsky I, McGeary SE, Bartel DP (2013) Beyond secondary structure: primary-sequence determinants license Pri-miRNA hairpins for processing. Cell 152:844–858CrossRefGoogle Scholar
  4. Bofill-De Ros X, Gu S (2016) Guidelines for the optimal design of miRNA-based shRNAs. Methods 103:157–166CrossRefGoogle Scholar
  5. Borchert GM, Lanier W, Davidson BL (2006) RNA polymerase III transcribes human microRNAs. Nat Struct Mol Biol 13:1097–1101CrossRefGoogle Scholar
  6. Buchman TG (2005) RNAi. Crit Care Med 33:S441–S443CrossRefGoogle Scholar
  7. Chung KH, Hart CC, Al-Bassam S et al (2006) Polycistronic RNA polymerase II expression vectors for RNA interference based on BIC/miR-155. Nucleic Acids Res 34:e53CrossRefGoogle Scholar
  8. Denli AM, Tops BBJ, Plasterk RHA et al (2004) Processing of primary microRNAs by the microprocessor complex. Nature 432:231–235CrossRefGoogle Scholar
  9. Elbashir SM, Lendeckel W, Tuschl T (2001) RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev 15:188–200CrossRefGoogle Scholar
  10. Fang W, Bartel DP (2015) The menu of features that define primary microRNAs and enable de novo design of microRNA genes. Mol Cell 60:131–145CrossRefGoogle Scholar
  11. Fellmann C, Hoffmann T, Sridhar V et al (2013) An optimized microRNA backbone for effective single-copy RNAi. Cell Rep 5:1704–1713CrossRefGoogle Scholar
  12. Gregory RI, Shiekhattar R (2005) MicroRNA biogenesis and cancer. Cancer Res 65:3509–3512CrossRefGoogle Scholar
  13. Griffiths-Jones S (2010) MiRBase: microRNA sequences and annotation. Curr Protoc Bioinform 1291–12910Google Scholar
  14. Grimm D, Streetz KL, Jopling CL et al (2006) Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Nature 441:537–541CrossRefGoogle Scholar
  15. Grishok A, Pasquinelli AE, Conte D et al (2001) Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell 106:23–34CrossRefGoogle Scholar
  16. Khan AA, Betel D, Miller ML et al (2009) Transfection of small RNAs globally perturbs gene regulation by endogenous microRNAs. Nat Biotechnol 27:549–555CrossRefGoogle Scholar
  17. Kim VN (2005) MicroRNA biogenesis: coordinated cropping and dicing. Nat Rev Mol Cell Biol 6:376–385CrossRefGoogle Scholar
  18. Lee Y, Ahn C, Han J et al (2003) The nuclear RNase III Drosha initiates microRNA processing. Nature 425:415–419CrossRefGoogle Scholar
  19. Liu J, Carmell MA, Rivas FV et al (2004) Argonaute2 is the catalytic engine of mammalian RNAi. Science 80-:305:1437–1441CrossRefGoogle Scholar
  20. Liu YP, Haasnoot J, ter Brake O et al (2008) Inhibition of HIV-1 by multiple siRNAs expressed from a single microRNA polycistron. Nucleic Acids Res 36:2811–2824CrossRefGoogle Scholar
  21. Mäkinen PI, Koponen JK, Kärkkäinen AM et al (2006) Stable RNA interference: comparison of U6 and H1 promoters in endothelial cells and in mouse brain. J Gene Med 8:433–441CrossRefGoogle Scholar
  22. Nakanishi Y, Oikawa M, Kumagai T et al (2005) Elastic property of Kondo semiconductor CeOs4Sb12. Phys B Condens Matter 359–361:907–909CrossRefGoogle Scholar
  23. Safe S (2013) RNA interference. Brenner’s Encycl Genet Second Ed 418:288–289Google Scholar
  24. Siolas D, Lerner C, Burchard J et al (2005) Synthetic shRNAs as potent RNAi triggers. Nat Biotechnol 23:227–231CrossRefGoogle Scholar
  25. Song H, Yang P-C (2010) Construction of shRNA lentiviral vector. N Am J Med Sci 598–601Google Scholar
  26. Song JJ, Smith SK, Hannon GJ, Joshua-Tor L (2004) Crystal structure of argonaute and its implications for RISC slicer activity. Science 80(305):1434–1437CrossRefGoogle Scholar
  27. Stegmeier F, Hu G, Rickles RJ et al (2005) A lentiviral microRNA-based system for single-copy polymerase II-regulated RNA interference in mammalian cells. Proc Natl Acad Sci 102:13212–13217CrossRefGoogle Scholar
  28. Tijsterman M, Plasterk RHA (2004) Dicers at RISC: the mechanism of RNAi. Cell 117:1–3CrossRefGoogle Scholar
  29. Van Gestel MA, Van Erp S, Sanders LE et al (2014) shRNA-induced saturation of the microRNA pathway in the rat brain. Gene Ther 21:205–211CrossRefGoogle Scholar
  30. Winter J, Jung S, Keller S et al (2009) Many roads to maturity: microRNA biogenesis pathways and their regulation. Nat Cell Biol 11:228–234CrossRefGoogle Scholar
  31. Xia XG, Zhou H, Ding H et al (2003) An enhanced U6 promoter for synthesis of short hairpin RNA. Nucleic Acids Res 31:e100CrossRefGoogle Scholar
  32. Zeng Y, Wagner EJ, Cullen BR (2002) Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells. Mol Cell 9:1327–1333CrossRefGoogle Scholar

Copyright information

© The Genetics Society of Korea 2019

Authors and Affiliations

  1. 1.Department of Molecular Bioscience, College of Biomedical ScienceKangwon National UniversityChuncheonRepublic of Korea
  2. 2.Division of Biomedical Convergence, College of Biomedical ScienceKangwon National UniversityChuncheonRepublic of Korea

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