Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Perspective
  • Published:

Enzymatically prepared RNAi libraries

Nature Methods volume 3, pages 696–700 (2006)Cite this article

Abstract

Large-scale RNA interference (RNAi) screens in mammalian cells have mainly used synthetic small interfering RNA (siRNA) or short hairpin RNA (shRNA) libraries. The RNAi triggers for both of these approaches were designed with algorithm-based predictions to identify single sequences for mRNA knockdown. Alternatives to these approaches have recently been developed using enzymatic methods. Here we describe the concepts of enzymatically prepared shRNA and siRNA libraries, and discuss their strengths and limitations.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Generation of siRNA pools by endoribonuclease cleavage.
Figure 2: Construction of shRNA libraries using the methods EPRIL, SPEED and REGS.

Similar content being viewed by others

References

  1. Elbashir, S.M. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498 (2001).

    Article  CAS  Google Scholar 

  2. Ketting, R.F. et al. Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans. Genes Dev. 15, 2654–2659 (2001).

    Article  CAS  Google Scholar 

  3. Elbashir, S.M., Lendeckel, W. & Tuschl, T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 15, 188–200 (2001).

    Article  CAS  Google Scholar 

  4. Chang, K., Elledge, S.J. & Hannon, G.J. Lessons from nature: microRNA-based shRNA libraries. Nat. Methods 3, 707–714 (2006).

    Article  CAS  Google Scholar 

  5. Bernards, R., Brummelkamp, T.R. & Beijersbergen, R.L. shRNA libraries and their use in cancer genetics. Nat. Methods 3, 701–706 (2006).

    Article  CAS  Google Scholar 

  6. Root, D.E., Hacohen, N., Hahn, W.C., Lander, E.S. & Sabatini, D.M. Genome-scale loss-of-function screening with a lentiviral RNAi library. Nat. Methods 3, 715–719 (2006).

    Article  CAS  Google Scholar 

  7. Reynolds, A. et al. Rational siRNA design for RNA interference. Nat. Biotechnol. 22, 326–330 (2004).

    Article  CAS  Google Scholar 

  8. Taxman, D.J. et al. Criteria for effective design, construction, and gene knockdown by shRNA vectors. BMC Biotechnol. 6, 7 (2006).

    Article  Google Scholar 

  9. Kittler, R. & Buchholz, F. RNA interference: gene silencing in the fast lane. Semin. Cancer Biol. 13, 259–265 (2003).

    Article  CAS  Google Scholar 

  10. Yang, D. et al. Short RNA duplexes produced by hydrolysis with Escherichia coli RNase III mediate effective RNA interference in mammalian cells. Proc. Natl. Acad. Sci. USA 99, 9942–9947 (2002).

    Article  CAS  Google Scholar 

  11. Myers, J.W., Jones, J.T., Meyer, T. & Ferrell, J.E., Jr. Recombinant Dicer efficiently converts large dsRNAs into siRNAs suitable for gene silencing. Nat. Biotechnol. 21, 324–328 (2003).

    Article  CAS  Google Scholar 

  12. Kawasaki, H., Suyama, E., Iyo, M. & Taira, K. siRNAs generated by recombinant human Dicer induce specific and significant but target site-independent gene silencing in human cells. Nucleic Acids Res. 31, 981–987 (2003).

    Article  CAS  Google Scholar 

  13. Buchholz, F., Drechsel, D., Ruer, M. & Kittler, R. Production of siRNA in vitro by enzymatic digestion of double-stranded RNA. in Gene Silencing by RNA: Technology and Application (ed., Sohail, M.) 87–99 (CRC Press, Boca Raton, 2004).

    Google Scholar 

  14. Yang, D., Goga, A. & Bishop, J.M. RNA interference (RNAi) with RNase III-prepared siRNAs. Methods Mol. Biol. 252, 471–482 (2004).

    CAS  PubMed  Google Scholar 

  15. Zhu, C. & Jiang, W. Cell cycle-dependent translocation of PRC1 on the spindle by Kif4 is essential for midzone formation and cytokinesis. Proc. Natl. Acad. Sci. USA 102, 343–348 (2005).

    Article  CAS  Google Scholar 

  16. Zhu, C. et al. Functional analysis of human microtubule-based motor proteins, the kinesins and dyneins, in mitosis/cytokinesis using RNA interference. Mol. Biol. Cell 16, 3187–3199 (2005).

    Article  CAS  Google Scholar 

  17. Hoepfner, S. et al. Modulation of receptor recycling and degradation by the endosomal kinesin KIF16B. Cell 121, 437–450 (2005).

    Article  CAS  Google Scholar 

  18. Schnatwinkel, C. et al. The Rab5 effector Rabankyrin-5 regulates and coordinates different endocytic mechanisms. PLoS Biol. 2, E261 (2004).

    Article  Google Scholar 

  19. Calegari, F., Haubensak, W., Yang, D., Huttner, W.B. & Buchholz, F. Tissue-specific RNA interference in postimplantation mouse embryos with endoribonuclease-prepared short interfering RNA. Proc. Natl. Acad. Sci. USA 99, 14236–14240 (2002).

    Article  CAS  Google Scholar 

  20. Calegari, F., Marzesco, A.M., Kittler, R., Buchholz, F. & Huttner, W.B. Tissue-specific RNA interference in post-implantation mouse embryos using directional electroporation and whole embryo culture. Differentiation 72, 92–102 (2004).

    Article  CAS  Google Scholar 

  21. Xuan, B., Qian, Z., Hong, J. & Huang, W. EsiRNAs inhibit Hepatitis B virus replication in mice model more efficiently than synthesized siRNAs. Virus Res. 118, 150–155 (2006).

    Article  CAS  Google Scholar 

  22. Kronke, J. et al. Alternative approaches for efficient inhibition of hepatitis C virus RNA replication by small interfering RNAs. J. Virol. 78, 3436–3446 (2004).

    Article  Google Scholar 

  23. Kittler, R., Heninger, A.K., Franke, K., Habermann, B. & Buchholz, F. Production of endoribonuclease-prepared short interfering RNAs for gene silencing in mammalian cells. Nat. Methods 2, 779–784 (2005).

    Article  CAS  Google Scholar 

  24. Myers, J.W. & Ferrell, J.E. Silencing gene expression with Dicer-generated siRNA pools. Methods Mol. Biol. 309, 93–196 (2005).

    CAS  PubMed  Google Scholar 

  25. Kittler, R. et al. An endoribonuclease-prepared siRNA screen in human cells identifies genes essential for cell division. Nature 432, 1036–1040 (2004).

    Article  CAS  Google Scholar 

  26. Liou, J. et al. STIM is a Ca2+ sensor essential for Ca2+-store-depletion-triggered Ca2+ influx. Curr. Biol. 15, 1235–1241 (2005).

    Article  CAS  Google Scholar 

  27. Henschel, A., Buchholz, F. & Habermann, B. DEQOR: a web-based tool for the design and quality control of siRNAs. Nucleic Acids Res. 32, W113–W120 (2004).

    Article  CAS  Google Scholar 

  28. Shirane, D. et al. Enzymatic production of RNAi libraries from cDNAs. Nat. Genet. 36, 190–196 (2004).

    Article  CAS  Google Scholar 

  29. Sen, G., Wehrman, T.S., Myers, J.W. & Blau, H.M. Restriction enzyme-generated siRNA (REGS) vectors and libraries. Nat. Genet. 36, 183–189 (2004).

    Article  CAS  Google Scholar 

  30. Luo, B., Heard, A.D. & Lodish, H.F. Small interfering RNA production by enzymatic engineering of DNA (SPEED). Proc. Natl. Acad. Sci. USA 101, 5494–5499 (2004).

    Article  CAS  Google Scholar 

  31. Seyhan, A.A. et al. Complete, gene-specific siRNA libraries: production and expression in mammalian cells. RNA 11, 837–846 (2005).

    Article  CAS  Google Scholar 

  32. Zhao, H.F. et al. High-throughput screening of effective siRNAs from RNAi libraries delivered via bacterial invasion. Nat. Methods 2, 967–973 (2005).

    Article  CAS  Google Scholar 

  33. Jackson, A.L. et al. Expression profiling reveals off-target gene regulation by RNAi. Nat. Biotechnol. 21, 635–637 (2003).

    Article  CAS  Google Scholar 

  34. Bridge, A.J., Pebernard, S., Ducraux, A., Nicoulaz, A.L. & Iggo, R. Induction of an interferon response by RNAi vectors in mammalian cells. Nat. Genet. 34, 263–264 (2003).

    Article  CAS  Google Scholar 

  35. Scacheri, P.C. et al. Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells. Proc. Natl. Acad. Sci. USA 101, 1892–1897 (2004).

    Article  CAS  Google Scholar 

  36. Jackson, A.L. & Linsley, P.S. Noise amidst the silence: off-target effects of siRNAs? Trends Genet. 20, 521–524 (2004).

    Article  CAS  Google Scholar 

  37. Lin, X. et al. siRNA-mediated off-target gene silencing triggered by a 7 nt complementation. Nucleic Acids Res. 33, 4527–4535 (2005).

    Article  CAS  Google Scholar 

  38. Birmingham, A. et al. 3′ UTR seed matches, but not overall identity, are associated with RNAi off-targets. Nat. Methods 3, 199–204 (2006).

    Article  CAS  Google Scholar 

  39. Jackson, A.L. et al. Widespread siRNA “off-target” transcript silencing mediated by seed region sequence complementarity. RNA 12, 1179–1187 (2006).

    Article  CAS  Google Scholar 

  40. Dorsett, Y. & Tuschl, T. siRNAs: applications in functional genomics and potential as therapeutics. Nat. Rev. Drug Discov. 3, 318–329 (2004).

    Article  CAS  Google Scholar 

  41. Kittler, R. & Buchholz, F. Functional genomic analysis of cell division by endoribonuclease-prepared siRNAs. Cell Cycle 4, 564–567 (2005).

    Article  CAS  Google Scholar 

  42. Bailey, S.N., Ali, S.M., Carpenter, A.E., Higgins, C.O. & Sabatini, D.M. Microarrays of lentiviruses for gene function screens in immortalized and primary cells. Nat. Methods 3, 117–122 (2006).

    Article  CAS  Google Scholar 

  43. Moffat, J. et al. A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell 124, 1283–1298 (2006).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank B. Habermann, V. Surendranath and E. Krausz for discussions. This work was supported by the EU grants “FunGenES” (LSHG-CT-2003-503494), “Mitocheck” (LSHG-CT-2004-503464), and by the Nationales Genomforschungsnetz 2 grant SMP-RNAi (01GR0402).

Author information

Authors and Affiliations

  1. Max Planck Institute for Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, Dresden, D-01307, Germany

    Frank Buchholz, Ralf Kittler, Mikolaj Slabicki & Mirko Theis

Authors
  1. Frank Buchholz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ralf Kittler
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mikolaj Slabicki
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mirko Theis
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Frank Buchholz.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Buchholz, F., Kittler, R., Slabicki, M. et al. Enzymatically prepared RNAi libraries. Nat Methods 3, 696–700 (2006). https://doi.org/10.1038/nmeth912

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth912

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing