GENE EXPRESSION SILENCING METHODS IN IN VITRO MODELS
DOI:
https://doi.org/10.31435/ijitss.1(49).2026.4984Keywords:
Gene Silencing, RNA Interference, CRISPRi, Antisense OligonucleotidesAbstract
Gene expression silencing is one of the key tools in molecular biology and is widely applied in in vitro studies to investigate gene function and the mechanisms regulating cellular processes. The dynamic development of molecular techniques has resulted in the availability of numerous strategies that enable either transient or permanent reduction of gene activity at different stages of gene expression. The aim of this work is to review and compare gene expression silencing methods used in in vitro models, with particular emphasis on their mechanisms of action, efficiency, durability of the effect, and experimental limitations.
The article discusses classical approaches based on RNA interference, including the use of siRNA, shRNA, and microRNA modulation, as well as strategies employing antisense oligonucleotides. Special attention is given to technologies based on the CRISPR system, including CRISPRi as a tool for reversible transcriptional repression and CRISPR–Cas9, which enables permanent disruption of gene function. Issues related to the delivery of molecular tools into cells, validation of silencing efficiency, and the importance of appropriate experimental controls are also addressed.
Analysis of the literature indicates that the choice of an appropriate method should be tailored to the research objective, the characteristics of the gene under investigation, and the planned duration of the experiment. Increasingly, a complementary approach—combining different gene silencing strategies is recommended, as it enhances the reliability of results obtained in in vitro studies.
References
Sioud, M. (2020). RNA and CRISPR interferences: Past, present, and future perspectives. Methods in Molecular Biology, 2115, 1–22. https://doi.org/10.1007/978-1-0716-0290-4_1
El-Sappah, A. H., Yan, K., Huang, Q., Islam, M. M., Li, Q., Wang, Y., Khan, M. S., Zhao, X., Mir, R. R., Li, J., El-Tarabily, K. A., & Abbas, M. (2021). Comprehensive mechanism of gene silencing and its role in plant growth and development. Frontiers in Plant Science, 12, Article 705249. https://doi.org/10.3389/fpls.2021.705249
Huntzinger, E., & Izaurralde, E. (2011). Gene silencing by microRNAs: Contributions of translational repression and mRNA decay. Nature Reviews Genetics, 12(2), 99–110. https://doi.org/10.1038/nrg2936
O'Brien, J., Hayder, H., Zayed, Y., & Peng, C. (2018). Overview of microRNA biogenesis, mechanisms of actions, and circulation. Frontiers in Endocrinology, 9, Article 402. https://doi.org/10.3389/fendo.2018.00402
Echeverri, C. J., & Perrimon, N. (2006). High-throughput RNAi screening in cultured cells: A user's guide. Nature Reviews Genetics, 7(5), 373–384. https://doi.org/10.1038/nrg1836
Boutros, M., & Ahringer, J. (2008). The art and design of genetic screens: RNA interference. Nature Reviews Genetics, 9(7), 554–566. https://doi.org/10.1038/nrg2364
Mohr, S., Bakal, C., & Perrimon, N. (2010). Genomic screening with RNAi: Results and challenges. Annual Review of Biochemistry, 79, 37–64. https://doi.org/10.1146/annurev-biochem-060408-092949
Housden, B. E., & Perrimon, N. (2016). Comparing CRISPR and RNAi-based screening technologies. Nature Biotechnology, 34(6), 621–623. https://doi.org/10.1038/nbt.3599
Wang, T., Birsoy, K., Hughes, N. W., Krupczak, K. M., Post, Y., Wei, J. J., Lander, E. S., & Sabatini, D. M. (2015). Identification and characterization of essential genes in the human genome. Science, 350(6264), 1096–1101. https://doi.org/10.1126/science.aac7041
Scadden, A. D., & Smith, C. W. (2001). RNAi is antagonized by A-->I hyper-editing. EMBO Reports, 2(12), 1107–1111. https://doi.org/10.1093/embo-reports/kve244
Echeverri, C. J., Beachy, P. A., Baum, B., Boutros, M., Buchholz, F., Chanda, S. K., Downward, J., Ellenberg, J., Fraser, A. G., Hacohen, N., Hahn, W. C., Jackson, A. L., Kiger, A., Linsley, P. S., Lum, L., Ma, Y., Mathey-Prévôt, B., Root, D. E., Sabatini, D. M., ... Bernards, R. (2006). Minimizing the risk of reporting false positives in large-scale RNAi screens. Nature Methods, 3(10), 777–779. https://doi.org/10.1038/nmeth1006-777
Jackson, A. L., & Linsley, P. S. (2010). Recognizing and avoiding siRNA off-target effects for target identification and therapeutic application. Nature Reviews Drug Discovery, 9(1), 57–67. https://doi.org/10.1038/nrd3010
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
Hammond, S. M., Boettcher, S., Caudy, A. A., Kobayashi, R., & Hannon, G. J. (2001). Argonaute2, a link between genetic and biochemical analyses of RNAi. Science, 293(5532), 1146–1150. https://doi.org/10.1126/science.1064023
Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., & Tuschl, T. (2001). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 411(6836), 494–498. https://doi.org/10.1038/35078107
Hannon, G. J. (2002). RNA interference. Nature, 418(6894), 244–251. https://doi.org/10.1038/418244a
Kim, D. H., & Rossi, J. J. (2007). Strategies for silencing human disease using RNA interference. Nature Reviews Genetics, 8(3), 173–184. https://doi.org/10.1038/nrg2006
Roberts, T. C., Langer, R., & Wood, M. J. A. (2020). Advances in oligonucleotide drug delivery. Nature Reviews Drug Discovery, 19(10), 673–694. https://doi.org/10.1038/s41573-020-0075-7
Bennett, C. F., & Swayze, E. E. (2010). RNA targeting therapeutics: Molecular mechanisms of antisense oligonucleotides as a therapeutic platform. Annual Review of Pharmacology and Toxicology, 50, 259–293. https://doi.org/10.1146/annurev.pharmtox.010909.105654
Rinaldi, C., & Wood, M. J. A. (2018). Antisense oligonucleotides: The next frontier for treatment of neurological disorders. Nature Reviews Neurology, 14(1), 9–21. https://doi.org/10.1038/nrneurol.2017.148
Crooke, S. T. (2017). Molecular mechanisms of antisense oligonucleotides. Nucleic Acid Therapeutics, 27(2), 70–77. https://doi.org/10.1089/nat.2016.0656
Singh, N. N., Luo, D., & Singh, R. N. (2018). Pre-mRNA splicing modulation by antisense oligonucleotides. Methods in Molecular Biology, 1828, 415–437. https://doi.org/10.1007/978-1-4939-8651-4_26
Havens, M. A., & Hastings, M. L. (2016). Splice-switching antisense oligonucleotides as therapeutic drugs. Nucleic Acids Research, 44(14), 6549–6563. https://doi.org/10.1093/nar/gkw533
Qi, L. S., Larson, M. H., Gilbert, L. A., Doudna, J. A., Weissman, J. S., Arkin, A. P., & Lim, W. A. (2013). Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell, 152(5), 1173–1183. https://doi.org/10.1016/j.cell.2013.02.022
Doudna, J. A., & Charpentier, E. (2014). Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science, 346(6213), Article 1258096. https://doi.org/10.1126/science.1258096
Gilbert, L. A., Larson, M. H., Morsut, L., Liu, Z., Brar, G. A., Torres, S. E., Stern-Ginossar, N., Brandman, O., Whitehead, E. H., Doudna, J. A., Lim, W. A., Weissman, J. S., & Qi, L. S. (2013). CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell, 154(2), 442–451. https://doi.org/10.1016/j.cell.2013.06.044
Dominguez, A. A., Lim, W. A., & Qi, L. S. (2016). Beyond editing: Repurposing CRISPR-Cas9 for precision genome regulation and interrogation. Nature Reviews Molecular Cell Biology, 17(1), 5–15. https://doi.org/10.1038/nrm.2015.2
Kearns, N. A., Pham, H., Tabak, B., Genga, R. M., Silverstein, N. J., Garber, M., & Maehr, R. (2015). Functional annotation of native enhancers with a Cas9-histone demethylase fusion. Nature Methods, 12(5), 401–403. https://doi.org/10.1038/nmeth.3325
Amabile, A., Migliara, A., Capasso, P., Biffi, M., Cittaro, D., Naldini, L., & Lombardo, A. (2016). Inheritable silencing of endogenous genes by hit-and-run targeted epigenetic editing. Cell, 167(1), 219–232.e14. https://doi.org/10.1016/j.cell.2016.09.006
Thakore, P. I., D'Ippolito, A. M., Song, L., Safi, A., Shivakumar, N. K., Kabadi, A. M., Reddy, T. E., Crawford, G. E., & Gersbach, C. A. (2015). Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements. Nature Methods, 12(12), 1143–1149. https://doi.org/10.1038/nmeth.3630
Doench, J. G., Fusi, N., Sullender, M., Hegde, M., Vaimberg, E. W., Donovan, K. F., Smith, I., Tothova, Z., Wilen, C., Orchard, R., Virgin, H. W., Listgarten, J., & Root, D. E. (2016). Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nature Biotechnology, 34(2), 184–191. https://doi.org/10.1038/nbt.3437
Horlbeck, M. A., Witkowsky, L. B., Guglielmi, B., Replogle, J. M., Gilbert, L. A., Villalta, J. E., Torigoe, S. E., Tjian, R., & Weissman, J. S. (2016). Nucleosomes impede Cas9 access to DNA in vivo and in vitro. eLife, 5, Article e12677. https://doi.org/10.7554/eLife.12677
Shalem, O., Sanjana, N. E., & Zhang, F. (2015). High-throughput functional genomics using CRISPR-Cas9. Nature Reviews Genetics, 16(5), 299–311. https://doi.org/10.1038/nrg3899
Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A., & Zhang, F. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science, 339(6121), 819–823. https://doi.org/10.1126/science.1231143
Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 337(6096), 816–821. https://doi.org/10.1126/science.1225829
Rossi, A., Kontarakis, Z., Gerri, C., Nolte, H., Hölper, S., Krüger, M., & Stainier, D. Y. (2015). Genetic compensation induced by deleterious mutations but not gene knockdowns. Nature, 524(7564), 230–233. https://doi.org/10.1038/nature14580
Kosicki, M., Tomberg, K., & Bradley, A. (2018). Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements. Nature Biotechnology, 36(8), 765–771. https://doi.org/10.1038/nbt.4192
Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott, D. A., & Zhang, F. (2013). Genome engineering using the CRISPR-Cas9 system. Nature Protocols, 8(11), 2281–2308. https://doi.org/10.1038/nprot.2013.143
Haapaniemi, E., Botla, S., Persson, J., Schmierer, B., & Taipale, J. (2018). CRISPR-Cas9 genome editing induces a p53-mediated DNA damage response. Nature Medicine, 24(7), 927–930. https://doi.org/10.1038/s41591-018-0049-z
Gilleron, J., Querbes, W., Zeigerer, A., Borodovsky, A., Marsico, G., Schubert, U., Manygoats, K., Seifert, S., Andree, C., Stöter, M., Epstein-Barash, H., Zhang, L., Koteliansky, V., Fitzgerald, K., Fava, E., Bickle, M., Kalaidzidis, Y., Akinc, A., Maier, M., & Zerial, M. (2013). Image-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escape. Nature Biotechnology, 31(7), 638–646. https://doi.org/10.1038/nbt.2612
Kim, T. K., & Eberwine, J. H. (2010). Mammalian cell transfection: The present and the future. Analytical and Bioanalytical Chemistry, 397(8), 3173–3178. https://doi.org/10.1007/s00216-010-3821-6
Stewart, M. P., Sharei, A., Ding, X., Sahay, G., Langer, R., & Jensen, K. F. (2016). In vitro and ex vivo strategies for intracellular delivery. Nature, 538(7624), 183–192. https://doi.org/10.1038/nature19764
Neumann, E., Schaefer-Ridder, M., Wang, Y., & Hofschneider, P. H. (1982). Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO Journal, 1(7), 841–845. https://doi.org/10.1002/j.1460-2075.1982.tb01257.x
Naldini, L. (1998). Lentiviruses as gene transfer agents for delivery to non-dividing cells. Current Opinion in Biotechnology, 9(5), 457–463. https://doi.org/10.1016/S0958-1669(98)80029-3
Moffat, J., Grueneberg, D. A., Yang, X., Kim, S. Y., Kloepfer, A. M., Hinkle, G., Piqani, B., Eisenhaure, T. M., Luo, B., Grenier, J. K., Carpenter, A. E., Foo, S. Y., Stewart, S. A., Stockwell, B. R., Hacohen, N., Hahn, W. C., Lander, E. S., Sabatini, D. M., Root, D. E. (2006). A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell, 124(6), 1283–1298. https://doi.org/10.1016/j.cell.2006.01.040
Vogel, C., & Marcotte, E. M. (2012). Insights into the regulation of protein abundance from proteomic and transcriptomic analyses. Nature Reviews Genetics, 13(4), 227–232. https://doi.org/10.1038/nrg3185
Bustin, S. A., Benes, V., Garson, J. A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M. W., Shipley, G. L., Vandesompele, J., & Wittwer, C. T. (2009). The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry, 55(4), 611–622. https://doi.org/10.1373/clinchem.2008.112797
Wang, Z., Gerstein, M., & Snyder, M. (2009). RNA-Seq: A revolutionary tool for transcriptomics. Nature Reviews Genetics, 10(1), 57–63. https://doi.org/10.1038/nrg2484
Taylor, S. C., & Posch, A. (2014). The design of a quantitative western blot experiment. BioMed Research International, 2014, Article 361590. https://doi.org/10.1155/2014/361590
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Copyright (c) 2026 Michał Dorota, Alicja Dorota, Wojciech Żywiec, Kacper Karaban, Nicole Maryniak, Jakub Rzeszutek
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