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Efficient and specific gene knockdown by small interfering RNAs produced in bacteria

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Abstract

Synthetic small interfering RNAs (siRNAs) are an indispensable tool to investigate gene function in eukaryotic cells1,2 and may be used for therapeutic purposes to knock down genes implicated in disease3. Thus far, most synthetic siRNAs have been produced by chemical synthesis. Here we present a method to produce highly potent siRNAs in Escherichia coli. This method relies on ectopic expression of p19, an siRNA-binding protein found in a plant RNA virus4,5. When expressed in E. coli, p19 stabilizes an 21-nt siRNA-like species produced by bacterial RNase III. When mammalian cells are transfected by them, siRNAs that were generated in bacteria expressing p19 and a hairpin RNA encoding 200 or more nucleotides of a target gene reproducibly knock down target gene expression by 90% without immunogenicity or off-target effects. Because bacterially produced siRNAs contain multiple sequences against a target gene, they may be especially useful for suppressing polymorphic cellular or viral genes.

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Figure 1: Ectopic p19 expression captures small RNAs in E. coli.
Figure 2: pro-siRNAs knock down target gene expression.
Figure 3: pro-siRNA–mediated knockdown of endogenous and viral gene expression in human cells.
Figure 4: pro-siRNA sequences and assessment of off-target effects.

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Change history

  • 22 March 2013

    In the version of this article initially published online, in Figure 4d, the label EGFPPFL vs NC siRNA has been corrected to read EGFPFL vs NC siRNA. The error has been corrected for the print, PDF and HTML versions of this article.

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Acknowledgements

The authors thank J. Carrington (Donald Danforth Plant Science Center) for the p19 clone, C. Zhang and G. Ruvkun (Massachusetts General Hospital) for L4440 plasmid and HT115(DE3) strain, S. Kushner (University of Georgia) for SK7622 strain, D. Higgins (Harvard Medical School) for pLIV-1 plasmid, and S. Ranjbar (Boston Children's Hospital) for HIV strains and cell lines. We thank L.M. Mazzola, J.M. Bybee and D.B. Munafo from New England Biolabs for assistance with RNA deep sequencing and Z. Ansara for technical help. We thank A. Hochschild (Harvard Medical School) for suggestions and critical reading of the manuscript and Lieberman Lab members for technical assistance, helpful discussions and comments on the manuscript. This work was supported by National Institutes of Health grant AI087431 (J.L.) and a GSK-IDI Alliance fellowship (L.H.).

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Authors and Affiliations

Authors

Contributions

L.H. and J.L. designed the experiments with advice from J.J., L.M., and P.D. J.J. and L.M. prepared p19 beads and RNA deep sequencing libraries. P.D. constructed E. coli mutant strains. E.K. performed siRNA comparison and macrophage transfection experiments. L.H. performed all other experiments. L.H. and J.L. wrote the paper.

Corresponding author

Correspondence to Judy Lieberman.

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Competing interests

J.J. and L.M. are employees of New England Biolabs, a company that sells deep sequencing kits, p19 and other proteins for RNA and DNA research.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–12 and Supplementary Tables 1–3 and 5–8 (PDF 1425 kb)

Supplementary Table 4

Significantly changed genes (XLSX 104 kb)

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Huang, L., Jin, J., Deighan, P. et al. Efficient and specific gene knockdown by small interfering RNAs produced in bacteria. Nat Biotechnol 31, 350–356 (2013). https://doi.org/10.1038/nbt.2537

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