An oldie but a goodie — a phrase used to refer to something that has fallen out of fashion but is still useful and most importantly, effective. In the research world, there is a myriad of tools this could be applied to. When it comes to altering gene expression, CRISPR technologies have gradually taken over as the preferred method for many labs. However, RNA interference (RNAi) remains steadfast in the background, a handy “oldie” as you might say.
RNA interference (RNAi) is, as the name might suggest, an RNA-mediated gene silencing mechanism. RNAi was a landmark discovery in the 1990s and quickly rose to popularity. Compared to CRISPR, RNAi methods are often quicker and the repositories of validated small RNAs are vast and difficult to beat in terms of size. Like CRISPR, there is a variety of small RNA types to choose from and can be catered for your specific experiment. For this blog, we’ll be focusing on shRNA, one of the most popular choices for gene silencing.
RNA interference
RNAi is a useful tool to investigate the roles of specific genes or to knock down genes with potentially harmful mutations in therapeutic settings. Silencing of a target gene is achieved through the delivery of a double-stranded RNA (dsRNA) that matches the mRNA target sequence. The dsRNA can be delivered as any type of small RNA and can occur via transfection or viral delivery of a plasmid.
RNAi is an umbrella term for a number of different applications that involve different types of small RNA molecules, such as:
- Micro-RNA (miRNA)
- Short hairpin RNA (shRNA)
- Short interfering RNA (siRNA)
- Piwi-interacting RNA (piRNA)
miRNA and piRNA can be found endogenously in many cell types, while shRNA and siRNA are synthetic constructs introduced through external methods.
shRNA processing
Following delivery into cells using viral vectors or plasmids, shRNAs hijack the endogenous miRNA machinery for processing (Figure 1). The shRNA sequence is transcribed to produce hairpin shRNA structures that are processed by a ribonuclease called Dicer. In a great simplification of the process, Dicer chops off the loop of hairpin structures to create a mature siRNA duplex.
Fun fact! Endogenous miRNA production starts with transcription of the miRNA to create pri-miRNA, which is then processed by a complex called Drosha to pre-miRNA. miRNA plasmids are engineered to skip this first pri-miRNA step.
These RNA duplexes bind to the RNA-induced silencing complex (RISC). After binding to RISC, the RNA duplex is processed into a single targeting strand, with the extra strand usually being degraded. The target strand binds to the mRNA, which is then cleaved by RISC and degraded. In the miRNA pathway, RISC doesn’t always cleave the mRNA — the bound miRISC can simply block translation machinery.
The composition of the RISC varies, but at the very minimum requires an Argonaute protein. This Argonaute protein is what binds the small RNA and either cleaves the target mRNA or recruits other silencing proteins.
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| Figure 1: Comparison of shRNA and miRNA processing and silencing pathways. Created on BioRender.com. |
Expressing shRNAs using plasmids
The design of shRNA plasmids requires three important steps:
- Designing the shRNA sequence
- Choosing the plasmid backbone
- Choosing the promoter
Mature siRNAs typically range from 19–22 nucleotides and are 100% complementary to their target sequence. However, you can’t just use the mature sequence in a plasmid and call it a day. You need to include both sides of the complementary RNA duplex and the hairpin loop sequence (Figure 2A). Since you don’t need additional flanking sequences, shRNA plasmids are a bit smaller, with the full sequence being around 60 base pairs. This is much shorter than miRNA plasmids, which require additional flanking regions comprised of upstream and downstream genomic sequences (Figure 2B).
When it comes to the backbone, shRNAs are most often expressed constitutively, either in lentiviral or retroviral backbones. On some occasions, shRNAs are transiently expressed using either AAV backbones or species-specific expression plasmids. You can also bypass Dicer processing by using synthetic, mature siRNA. siRNAs are delivered via transient transfections and are handy for quick in vitro experiments.
shRNA plasmids typically use RNA polymerase III promoters, which transcribe small RNAs. The most common promoters are U6, H1, and 7SK. These are strong promoters with no tissue specificity, so in some cases they can lead to toxicity and off target effects. More recently, shRNA plasmids are being designed using RNA polymerase II promoters. The design of these plasmids is meant to more closely mimic pre-miRNAs, and the use of the pol II promoters allows for more flexibility in expression levels and tissue specificity.
Using your shRNA plasmids
One of the most common uses of shRNAs is to generate stable knockout or knockdown cell lines. In fact, it was the go-to choice for many years before CRISPR burst on to the scene. shRNAs reliably knock down a target gene but have a lot of off-target effects. However, this is a common issue with many gene silencing methods and just needs to be taken into account when designing experiments.
Interfering in the future
While RNAi has retreated a bit to the background, it remains a solid option for altering the expression of specific mRNAs. shRNAs are a quick way to create stable knockdown cells lines, and you can usually find validated shRNA sequences for your intended target. RNAi is a valuable tool for your research arsenal, and there is still a dedicated field of study for improving these tools. Next time you need to knock down a gene, maybe you’ll consider using RNAi!
References and Resources
References
Bartel, D. P. (2004). MicroRNAs. Cell, 116(2), 281–297. https://doi.org/10.1016/s0092-8674(04)00045-5
De Rie, D., Abugessaisa, I., Alam, T., Arner, E., Arner, P., Ashoor, H., Åström, G., Babina, M., Bertin, N., Burroughs, A. M., Carlisle, A. J., Daub, C. O., Detmar, M., Deviatiiarov, R., Fort, A., Gebhard, C., Goldowitz, D., Guhl, S., Ha, T. J., . . . De Hoon, M. J. L. (2017). An integrated expression atlas of miRNAs and their promoters in human and mouse. Nature Biotechnology, 35(9), 872–878. https://doi.org/10.1038/nbt.3947
Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., & Mello, C. C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 391(6669), 806–811. https://doi.org/10.1038/35888
Iwakawa, H., & Tomari, Y. (2021). Life of RISC: Formation, action, and degradation of RNA-induced silencing complex. Molecular Cell, 82(1), 30–43. https://doi.org/10.1016/j.molcel.2021.11.026
Kilikevicius, A., Meister, G., & Corey, D. R. (2021). Reexamining assumptions about miRNA-guided gene silencing. Nucleic Acids Research, 50(2), 617–634. https://doi.org/10.1093/nar/gkab1256
Lee, Y., Kim, M., Han, J., Yeom, K., Lee, S., Baek, S. H., & Kim, V. N. (2004). MicroRNA genes are transcribed by RNA polymerase II. The EMBO Journal, 23(20), 4051–4060. https://doi.org/10.1038/sj.emboj.7600385
McIntyre, G. J., & Fanning, G. C. (2006). Design and cloning strategies for constructing shRNA expression vectors. BMC Biotechnology, 6(1). https://doi.org/10.1186/1472-6750-6-1
O’Brien, J., Hayder, H., Zayed, Y., & Peng, C. (2018). Overview of MicroRNA biogenesis, mechanisms of actions, and circulation. Frontiers in Endocrinology, 9. https://doi.org/10.3389/fendo.2018.00402
Pratt, A. J., & MacRae, I. J. (2009). The RNA-induced Silencing Complex: a versatile gene-silencing machine. Journal of Biological Chemistry, 284(27), 17897–17901. https://doi.org/10.1074/jbc.r900012200
Ros, X. B., & Ørom, U. a. V. (2023). Recent progress in miRNA biogenesis and decay. RNA Biology, 21(1), 36–43. https://doi.org/10.1080/15476286.2023.2288741
Taxman, D. J., Moore, C. B., Guthrie, E. H., & Huang, M. T. (2010). Short hairpin RNA (SHRNA): Design, delivery, and assessment of gene knockdown. Methods in Molecular Biology, 139–156. https://doi.org/10.1007/978-1-60761-657-3_10
Vergani-Junior, C. A., Tonon-Da-Silva, G., Inan, M. D., & Mori, M. A. (2021). DICER: structure, function, and regulation. Biophysical Reviews, 13(6), 1081–1090. https://doi.org/10.1007/s12551-021-00902-w
Additional resources on the Addgene blog
- RNA Interference in Plant Biology: New Tools for an Old Favorite
- CRISPR 101: Targeting RNA with Cas13a (C2c2)
- Plasmids 101: The Promoter Region – Let's Go!
- Browse All Plasmids 101 Posts
Additional resources on addgene.org
Topics: Plasmids 101, Other Plasmid Tools, Plasmids
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