Second-generation shRNA libraries covering the mouse and human genomes

Abstract

Loss-of-function phenotypes often hold the key to understanding the connections and biological functions of biochemical pathways. We and others previously constructed libraries of short hairpin RNAs that allow systematic analysis of RNA interference–induced phenotypes in mammalian cells. Here we report the construction and validation of second-generation short hairpin RNA expression libraries designed using an increased knowledge of RNA interference biochemistry. These constructs include silencing triggers designed to mimic a natural microRNA primary transcript, and each target sequence was selected on the basis of thermodynamic criteria for optimal small RNA performance. Biochemical and phenotypic assays indicate that the new libraries are substantially improved over first-generation reagents. We generated large-scale-arrayed, sequence-verified libraries comprising more than 140,000 second-generation short hairpin RNA expression plasmids, covering a substantial fraction of all predicted genes in the human and mouse genomes. These libraries are available to the scientific community.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Design and structure of shRNA-mir cassettes.
Figure 2: Construction of the second-generation library.
Figure 3: Validation of the second-generation library.
Figure 4: Performance of the second-generation library in a small-scale high-throughput screen.

References

  1. 1

    Nakayashiki, H. et al. RNA silencing as a tool for exploring gene function in ascomycete fungi. Fungal Genet. Biol. 42, 275–283 (2005).

    CAS  Article  Google Scholar 

  2. 2

    Tang, G. & Galili, G. Using RNAi to improve plant nutritional value: from mechanism to application. Trends Biotechnol. 22, 463–469 (2004).

    CAS  Article  Google Scholar 

  3. 3

    Dasgupta, R. & Perrimon, N. Using RNAi to catch Drosophila genes in a web of interactions: insights into cancer research. Oncogene 23, 8359–8365 (2004).

    CAS  Article  Google Scholar 

  4. 4

    Fraser, A. Towards full employment: using RNAi to find roles for the redundant. Oncogene 23, 8346–8352 (2004).

    CAS  Article  Google Scholar 

  5. 5

    Silva, J., Chang, K., Hannon, G.J. & Rivas, F.V. RNA-interference-based functional genomics in mammalian cells: reverse genetics coming of age. Oncogene 23, 8401–8409 (2004).

    CAS  Article  Google Scholar 

  6. 6

    Bartel, D.P. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297 (2004).

    CAS  Article  Google Scholar 

  7. 7

    He, L. & Hannon, G.J. MicroRNAs: small RNAs with a big role in gene regulation. Nat. Rev. Genet. 5, 522–531 (2004).

    CAS  Article  Google Scholar 

  8. 8

    Reinhart, B.J. et al. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 403, 901–906 (2000).

    CAS  Article  Google Scholar 

  9. 9

    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).

    CAS  Article  Google Scholar 

  10. 10

    Grishok, A. et al. Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell 106, 23–34 (2001).

    CAS  Article  Google Scholar 

  11. 11

    Knight, S.W. & Bass, B.L. A role for the RNase III enzyme DCR-1 in RNA interference and germ line development in Caenorhabditis elegans. Science 293, 2269–2271 (2001).

    CAS  Article  Google Scholar 

  12. 12

    Hutvagner, G. et al. A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA. Science 293, 834–838 (2001).

    CAS  Article  Google Scholar 

  13. 13

    Lee, Y. et al. MicroRNA genes are transcribed by RNA polymerase II. EMBO J. 23, 4051–4060 (2004).

    CAS  Article  Google Scholar 

  14. 14

    Cai, X., Hagedorn, C.H. & Cullen, B.R. Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs. RNA 10, 1957–1966 (2004).

    CAS  Article  Google Scholar 

  15. 15

    Lee, Y. et al. The nuclear RNase III Drosha initiates microRNA processing. Nature 425, 415–419 (2003).

    CAS  Article  Google Scholar 

  16. 16

    Denli, A.M., Tops, B.B., Plasterk, R.H., Ketting, R.F. & Hannon, G.J. Processing of primary microRNAs by the Microprocessor complex. Nature 432, 231–235 (2004).

    CAS  Article  Google Scholar 

  17. 17

    Landthaler, M., Yalcin, A. & Tuschl, T. The human DiGeorge syndrome critical region gene 8 and its D. melanogaster homolog are required for miRNA biogenesis. Curr. Biol. 14, 2162–2167 (2004).

    CAS  Article  Google Scholar 

  18. 18

    Han, J. et al. The Drosha-DGCR8 complex in primary microRNA processing. Genes Dev. 18, 3016–3027 (2004).

    CAS  Article  Google Scholar 

  19. 19

    Gregory, R.I. et al. The Microprocessor complex mediates the genesis of microRNAs. Nature 432, 235–240 (2004).

    CAS  Article  Google Scholar 

  20. 20

    Yi, R., Qin, Y., Macara, I.G. & Cullen, B.R. Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev. 17, 3011–3016 (2003).

    CAS  Article  Google Scholar 

  21. 21

    Lund, E., Guttinger, S., Calado, A., Dahlberg, J.E. & Kutay, U. Nuclear export of microRNA precursors. Science 303, 95–98 (2004).

    CAS  Article  Google Scholar 

  22. 22

    Siolas, D. et al. Synthetic shRNAs as potent RNAi triggers. Nat. Biotechnol. 23, 227–231 (2005).

    CAS  Article  Google Scholar 

  23. 23

    Song, J.J. et al. The crystal structure of the Argonaute2 PAZ domain reveals an RNA binding motif in RNAi effector complexes. Nat. Struct. Biol. 10, 1026–1032 (2003).

    CAS  Article  Google Scholar 

  24. 24

    Schwarz, D.S. et al. Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199–208 (2003).

    CAS  Article  Google Scholar 

  25. 25

    Khvorova, A., Reynolds, A. & Jayasena, S.D. Functional siRNAs and miRNAs exhibit strand bias. Cell 115, 209–216 (2003).

    CAS  Article  Google Scholar 

  26. 26

    Paddison, P.J. et al. A resource for large-scale RNA-interference-based screens in mammals. Nature 428, 427–431 (2004).

    CAS  Article  Google Scholar 

  27. 27

    Berns, K. et al. A large-scale RNAi screen in human cells identifies new components of the p53 pathway. Nature 428, 431–437 (2004).

    CAS  Article  Google Scholar 

  28. 28

    Zeng, Y., Wagner, E.J. & Cullen, B.R. Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells. Mol. Cell 9, 1327–1333 (2002).

    CAS  Article  Google Scholar 

  29. 29

    Paddison, P.J., Caudy, A.A., Bernstein, E., Hannon, G.J. & Conklin, D.S. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev. 16, 948–958 (2002).

    CAS  Article  Google Scholar 

  30. 30

    Westbrook, T.F. et al. A genetic screen for candidate tumor suppressors identifies REST. Cell 121, 837–848 (2005).

    CAS  Article  Google Scholar 

  31. 31

    Chen, C.Z., Li, L., Lodish, H.F. & Bartel, D.P. MicroRNAs modulate hematopoietic lineage differentiation. Science 303, 83–86 (2004).

    CAS  Article  Google Scholar 

  32. 32

    Zeng, Y. & Cullen, B.R. Sequence requirements for micro RNA processing and function in human cells. RNA 9, 112–123 (2003).

    CAS  Article  Google Scholar 

  33. 33

    Kawasaki, H. & Taira, K. Short hairpin type of dsRNAs that are controlled by tRNA(Val) promoter significantly induce RNAi-mediated gene silencing in the cytoplasm of human cells. Nucleic Acids Res. 31, 700–707 (2003).

    CAS  Article  Google Scholar 

  34. 34

    Brummelkamp, T.R., Bernards, R. & Agami, R. A system for stable expression of short interfering RNAs in mammalian cells. Science 296, 550–553 (2002).

    CAS  Article  Google Scholar 

  35. 35

    Zheng, L. et al. An approach to genomewide screens of expressed small interfering RNAs in mammalian cells. Proc. Natl. Acad. Sci. USA 101, 135–140 (2004).

    CAS  Article  Google Scholar 

  36. 36

    Dickins, R.A. et al. Probing tumor phenotypes using stable and regulated synthetic microRNA precursors. Nat. Genet., advance online publication XX XXX 2005 (10.1038/ngXXX). [date and doi for lowe]

  37. 37

    Stegmeier, F., Hu, G., Rickles, R.J., Hannon, G.J. & Elledge, S.J. A lentiviral microRNA-based system for single copy Pol II regulated RNAi in mammalian cells. Proc. Natl. Acad. Sci. USA 102, 13212–13217 (2005).

    CAS  Article  Google Scholar 

  38. 38

    Li, M.Z. & Elledge, S.J. MAGIC, an in vivo genetic method for the rapid construction of recombinant DNA molecules. Nat. Genet. 37, 311–319 (2005).

    CAS  Article  Google Scholar 

  39. 39

    Cleary, M.A. et al. Production of complex nucleic acid libraries using highly parallel in situ oligonucleotide synthesis. Nat. Methods 1, 241–248 (2004).

    CAS  Article  Google Scholar 

  40. 40

    Li, X. et al. Generation of destabilized green fluorescent protein as a transcription reporter. J. Biol. Chem. 273, 34970–34975 (1998).

    CAS  Article  Google Scholar 

  41. 41

    Carmell, M.A. & Hannon, G.J. RNase III enzymes and the initiation of gene silencing. Nat. Struct. Mol. Biol. 11, 214–218 (2004).

    CAS  Article  Google Scholar 

  42. 42

    Elledge, S.J. & Walker, G.C. Phasmid vectors for identification of genes by complementation of Escherichia coli mutants. J. Bacteriol. 162, 777–783 (1985).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43

    Datsenko, K.A. & Wanner, B.L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97, 6640–6645 (2000).

    CAS  Article  Google Scholar 

  44. 44

    Cherepanov, P.P. & Wackernagel, W. Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. Gene 158, 9–14 (1995).

    CAS  Article  Google Scholar 

  45. 45

    Chalker, A.F., Leach, D.R. & Lloyd, R.G. Escherichia coli sbcC mutants permit stable propagation of DNA replicons containing a long palindrome. Gene 71, 201–205 (1988).

    CAS  Article  Google Scholar 

  46. 46

    Caudy, A.A., Myers, M., Hannon, G.J. & Hammond, S.M. Fragile X-related protein and VIG associate with the RNA interference machinery. Genes Dev. 16, 2491–2496 (2002).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank members of the laboratories of G.J.H., S.J.E. and S.W. Lowe for suggestions; J. Magnus for assistance with array PCRs; L. Nascimento, V. Balija, M. Kramer, T. Zutavern, S. Muller and B. Miller for assistance with sequencing; T. Moore for help with curation of the collection; and P. Linsley and S. Friend for their support of this project. This work was funded in part by awards from the Department of Defense Breast Cancer Research Program (G.J.H. and S.J.E.) and the US National Institutes of Health (G.J.H. and S.J.E.). S.J.E. and G.J.H. are investigators of the Howard Hughes Medical Institute.

Author information

Affiliations

Authors

Corresponding authors

Correspondence to Stephen J Elledge or Gregory J Hannon.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Mapping of Dicer and Drosha cleavage sites. (PDF 16 kb)

Supplementary Fig. 2

The complete insert sequences for pSM1 and pSM2 containing a luciferase shRNA are shown along with their most stable potential secondary structures as predicted by RNA fold. (PDF 100 kb)

Supplementary Fig. 3

Stable suppression by pSM2. (PDF 212 kb)

Supplementary Table 1

ShRNAs used in Figure 3. (PDF 86 kb)

Supplementary Table 2

ShRNAs used in Figure 4. (PDF 41 kb)

Supplementary Table 3

Oligonucleotides used in construction of the library vectors. (PDF 17 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Silva, J., Li, M., Chang, K. et al. Second-generation shRNA libraries covering the mouse and human genomes. Nat Genet 37, 1281–1288 (2005). https://doi.org/10.1038/ng1650

Download citation

Further reading

Search

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