Abstract
Basic biological research and biomedical applications often require studying the multiple interactions between genes or proteins while multiplex RNA interference (RNAi) technology is still challenging in mammalian cells. In mammalian genomes, the natural microRNA (miRNA) clusters, of which the miRNAs often share similar expression patterns and target diverse genes, would provide a potential multiplex RNAi scaffold. Based on the natural pri-miR-155 precursor, we have developed and characterized a multiplex RNAi method by engineering synthetic miRNA clusters, among which the maturation and function of individual miRNA precursors are independent of their positions in the cluster. And the synthetic miRNA clusters are assembled by an efficient hierarchical Golden-Gate cloning method. Here, we describe the design rules and the hierarchical cloning methods to construct synthetic miRNA cluster, and the brief protocol for the integration of synthetic miRNA clusters into the mammalian genome.
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References
Barabási A-L, Oltvai ZN (2004) Network biology: understanding the cell’s functional organization. Nat Rev Genet 5:101–113
Huttlin EL, Bruckner RJ, Paulo JA et al (2017) Architecture of the human interactome defines protein communities and disease networks. Nature 545:505–509
Kemp S, Wei H-M, Lu J-F et al (1998) Gene redundancy and pharmacological gene therapy: implications for X-linked adrenoleukodystrophy. Nat Med 4:1261–1268
Loscalzo J, Kohane I, Barabasi A-L (2007) Human disease classification in the postgenomic era: a complex systems approach to human pathobiology. Mol Syst Biol 3:124
Hu JX, Thomas CE, Brunak S (2016) Network biology concepts in complex disease comorbidities. Nat Rev Genet 17:615–629
Luo J, Emanuele MJ, Li D et al (2009) A genome-wide RNAi screen identifies multiple synthetic lethal interactions with the Ras oncogene. Cell 137:835–848
Mann KM, Ying H, Juan J et al (2016) KRAS-related proteins in pancreatic cancer. Pharmacol Ther 168:29–42
Gill H, Leung AYH, Kwong Y-L (2016) Molecularly targeted therapy in acute myeloid leukemia. Future Oncol 12:827–838
Martin SE, Jones TL, Thomas CL et al (2007) Multiplexing siRNAs to compress RNAi-based screen size in human cells. Nucleic Acids Res 35:e57
Mcintyre GJ, Yu Y-H, Tran A et al (2009) Cassette deletion in multiple shRNA lentiviral vectors for HIV-1 and its impact on treatment success. Virol J 6:184
Applegate TL, Birkett DJ, Mcintyre GJ et al (2010) In silico modeling indicates the development of HIV-1 resistance to multiple shRNA gene therapy differs to standard antiretroviral therapy. Retrovirology 7:83
Liu YP, Haasnoot J, Berkhout B (2007) Design of extended short hairpin RNAs for HIV-1 inhibition. Nucleic Acids Res 35:5683–5693
Sano M, Li H, Nakanishi M et al (2008) Expression of long anti-HIV-1 hairpin RNAs for the generation of multiple siRNAs: advantages and limitations. Mol Ther 16:170–177
Saayman SM, Arbuthnot P, Weinberg MS (2010) Effective pol III-expressed long hairpin RNAs targeted to multiple unique sites of HIV-1. Methods Mol Biol 629:159–174
Weinberg MS, Ely A, Barichievy S et al (2007) Specific inhibition of HBV replication in vitro and in vivo with expressed long hairpin RNA. Mol Ther 15:534–541
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 36:2811–2824
Aagaard LA, Zhang J, von Eije KJ et al (2008) Engineering and optimization of the miR-106b cluster for ectopic expression of multiplexed anti-HIV RNAs. Gene Ther 15:1536–1549
Israsena N, Supavonwong P, Ratanasetyuth N et al (2009) Inhibition of rabies virus replication by multiple artificial microRNAs. Antivir Res 84:76–83
ter Brake O, Berkhout B (2005) A novel approach for inhibition of HIV-1 by RNA interference: counteracting viral escape with a second generation of siRNAs. J RNAi Gene Silencing 1:56–65
Chung K-H, 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:e53
Wang T, Xie Y, Tan A et al (2016) Construction and characterization of a synthetic MicroRNA cluster for multiplex RNA interference in mammalian cells. ACS Synth Biol 5:1193–1200
Gong W, Ren Y, Zhou H et al (2008) siDRM: an effective and generally applicable online siRNA design tool. Bioinformatics 24:2405–2406
Ren Y, Gong W, Zhou H et al (2009) siRecords: a database of mammalian RNAi experiments and efficacies. Nucleic Acids Res 37:D146–D149
Snøve O, Nedland M, Fjeldstad SH et al (2004) Designing effective siRNAs with off-target control. Biochem Biophys Res Commun 325:769–773
Gabant P, Szpirer CY, Couturier M et al (1998) Direct selection cloning vectors adapted to the genetic analysis of gram-negative bacteria and their plasmids. Gene 207:87–92
Acknowledgments
We thank members of Xie lab for helpful discussions and useful suggestions. The research is supported by National Natural Science Foundation of China, NO: 31471255.
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Wang, T., Xie, Z. (2018). Construction and Integration of a Synthetic MicroRNA Cluster for Multiplex RNA Interference in Mammalian Cells. In: Braman, J. (eds) Synthetic Biology. Methods in Molecular Biology, vol 1772. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7795-6_19
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DOI: https://doi.org/10.1007/978-1-4939-7795-6_19
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