The discovery of RNA interference (RNAi) and its utilization in downregulation of specific target transcripts have revolutionized gene function analysis and elucidation of many key biochemical/genetic pathways. The insights into gene function, combined with a technology that made silencing of gene function possible using the potent, highly specific and selective RNAi approaches, provided the solution to longstanding complex obstacles in targeted crop improvementsfor agriculture, and disease therapies for medicine. In this introductory chapter, I aim briefly to cover the basics and peculiarities of RNAi and the advances made in understanding the mechanisms, components, function, evolution, application, safety and risk assessment of RNAi, while at the same time highlighting the related chapters of this book.
- Gene silencing
- RNA interference
- RNAi inducers and delivery
- RNAi-based disease therapy
The “central dogma” of genetics as first presented by Francis Crick is that genes, packed inside the cell as the deoxyribonucleic acid (DNA) molecule, are transcribed into messenger ribonucleic acids (mRNA), which are subsequently translated into proteins (or enzymes). These final protein products provide all life functions, and together with DNA and RNA, constitute the molecules of life. Therefore, if there is a disruption (interference) of a gene function, messenger RNA synthesis, or protein translation, normal life processes get altered or even stopped. “No gene-no messenger”, or “no messenger-no protein”, has been the basis of understanding biological processes. One of the easy-to-access points in cellular processes is messenger RNA due to its cytoplasmic location, “naked” structure, comparatively short half-life, and temporal existence between transcription and translation. Further, mRNA is in between the chain of events from DNA to protein; it has the universal chemical structure, consisting of only four nucleotides, regardless of the encoded message. In contrast, proteins are chemically much more variable, consisting of combinations of 21 different amino acids, with side chains that vary from very hydrophilic to highly hydrophobic. If mRNA is altered or eliminated before translation, there is no functional gene product, which results in changing the cellular process from the native state. This is the entire rationale of RNA interference (RNAi).
RNA interference is a process in eukaryotic cells in which double stranded endogenous or exogenous RNA molecules trigger a cytoplasmic response, which involves sequence specific target identification and destruction. This may include native messenger RNAs (mRNAs) that code vitally important proteins . Any type of double-stranded RNA (dsRNA) molecules can activate RNAi where long dsRNAs, microRNAs (miRNAs) and small interfering RNAs (siRNAs) and their various forms and modifications are considered the main players/inducers . Let us take a look at RNAi discovery history.
Plant scientists in the 1990s first used targeted gene silencing by introducing an antisense gene into plants. The first example was silencing of a nopaline synthase (NOS) gene, for which the silencing was only visible by loss of a band on a Norther blot and loss of NOS activity . The second antisense gene used in plants targeted the petunia chalcone synthase (CHS) gene, encoding the first step in floral pigment production, and the result was visible in the loss of petal pigmentation . Curiously, attempts to create dark pigmented petunia flowers by overexpression of the same CHS gene resulted in similar colorless petunia petals [4, 5]. It was thought that such a phenotype was “
Following these seminal discoveries, similar phenomena were discovered in other organisms including the nematode (
In plants, the suppression of targeted genes during viral infections was discovered  and subsequently developed into a system by which plant gene function may be studied through inhibition by infection with viruses bearing a short sequence targeted against plant mRNAs . This phenomenon was termed as "
Over the past decade, RNAi has been demonstrated in many eukaryotes including humans as well as some prokaryotic life forms  and has been recognized to form an integral part of many gene regulatory networks during development. This revolutionary breakthrough in biological science has become a valuable
RNAi research has rapidly advanced and expanded over the past decade, evidenced by increasing numbers of publications, research projects, and practical applications in both agriculture and medicine. For example, searching
2. Components, mechanism, and function
The principle mechanism of RNAi is complex, but very straightforward and easy to understand. RNAi is induced by the introduction of specific exogenous dsRNA either by virus genome RNAs, injection of synthetic dsRNAs or, in plants, is mediated by Agrobacterium. RNAi is also part of the normal development and dsRNAs are produced by endogenous genes encoding miRNA precursors or other long dsRNA molecules. In either case, the dsRNAs are recognized by the enzyme dicer and cleaved into short, double-stranded fragments of ~19-25 base pair long siRNAs . These siRNAs are separated into two single-stranded RNAs (ssRNAs), which are referred to as the “
It is not clear as yet how the
Although the pathways toward RNAi from exogenous and endogenous dsRNA converge at the RISC and use the same downstream RNAi machinery, there are also some clear differences in their processing and handling . Endogenous dsRNAs cleaved by dicer (
The main biological function of RNAi is regulation of gene activity of cells at the post-transcriptional level (PTGS) either by the inhibition of translation of mRNA or by direct degradation of the mRNA. In addition to PTGS, RNAi pathway components may contribute to maintenance of genome organization and structure, mediated by RNA-induced histone modification. Histone modification in turn affects heterochromatin formation and may silence gene activity at the pre-transcriptional level . This process is referred to as “RNA-induced gene silencing (RITS) and requires dicer, siRNA and RISC component proteins such as AGO and R2D2 . In addition, RNAi components and inducers (siRNA/dicer/AGO) may also possibly upregulate expression of genes in binding into a promoter region and through histone demethylation, a process dubbed RNA activation [37, 38].
Because of sequence-specific recognition, regulatory properties, and the possibility of systemic spreading of dsRNAs, RNAi is the key “sterilizing agent” of cells and tissues, and it functions as potent immune response against foreign nucleic acids from viruses, transposons, or transformation events which can invade and harm the genome and its stability . The chapters presented in Section 2 of this book have a more detailed coverage of the history of the RNAi discovery, mechanism, and functional components and on the biological role of RNAi including natural small RNAs/microRNAs as well as long noncoding RNAs in gene regulations.
3. Differences among organisms
Although the RNAi pathway is a universal process in eukaryotic cells, and it consists of similar component(s), mechanisms, and functions as described above, there are some variations among organisms in both up-take of exogenous dsRNAs and induction of RNAi. First, RNAi is systemic and heritable in plants and
RNAi is not found in some eukaryotic protozoa (e.g.,
Studies on components, mechanisms, and functions of RNAi have demonstrated variations among organisms, differences in eukaryotes and prokaryotes, and indicate that RNAi is derived from an ancestral immune defense function against transcripts of transposons and viruses [50, 51]. Although some eukaryotes might have lost RNAi components or, even, the entire pathway following the emergence of the Eukaryota, parsimony-based phylogenetic analyses suggest that an ancestral lineage of all eukaryotes possibly had a primitive RNAi capability including relevant components for some key functions such as histone modification . Phylogenetic studies also indicate that miRNAs of plants and animals may have evolved independently, but the conservation of some key proteins involved in RNAi also indicate that the last common ancestor of modern eukaryotes already possessed an siRNA-based gene silencing system. The RNAi-like defense system of prokaryotes is functionally similar, but structurally distinct from the eukaryotic RNAi system . It seems likely that a proto-RNAi system possessed at least some form of dicer-like, AGO, PIWI, and RdRP proteins. These basic components were shared by major eukaryotic lineages and functioned within an RNA degradation exosome complex .
Being an important component of an antiviral innate immune defense system in eukaryotes, RNAi components and various interaction/regulatory mechanisms, including the miRNA pathway, evolved later but at faster rates under strong directional selection . This could have been a means of generating an improved response to the evolutionary arms race with viral genes. Correspondingly, some plant viruses have evolved the means to suppress the RNAi response in their host cells . Extensive studies reported that an ancient duplication of RNAi components followed by species-specific gene duplications and losses provided evolutionary diversification, specificity and adaptation of the RNAi system in many organisms . Chapter(s) presented in Section 2 has covered some evolutionary aspects of RNAi.
Since its first discovery as anti-sense gene suppression,
The results of the functional genomics studies, advances in the understanding of the RNAi mechanism, improved design of trait-specific RNAi inducers (such as miRNAs), selection of target gene sequences combined with the development of proper delivery systems, as well as screens for “off-target” and cross reactivity have brought the practical applications of RNAi far beyond its initial experimental reach.
Agricultural application of RNAi through tissue culture-derived genetic modifications and transgenic research in a wide range of technical, food, and horticulture crops have been particularly successful and have solved many problems. Examples include, but are not limited to, crop yield and quality improvements [15, 58], food/nutrient quality improvements and fortification [59–62], decreasing the harmful precursors and carcinogens [63, 64], and improvement of plant pest and disease resistance [65–66]. Many of these applications are now evaluated for commercialization or are already in commercial production . In this context, targeting far red (FR) photoreceptor gene (
Therapeutic application of RNAi has also been successful in medicine and molecular pharmacology with examples in inflammatory and infectious disease [68-71], cancer [72-75], as well as hereditary and neurodegenerative diseases . Indeed, for many other disorders RNAi may have great potential. To highlight advances made on this field, in Section 4 of this book, we present several relevant chapters on advances of RNAi application in key human diseases of blood, ocular, nervous, kidney, and oncogenic origin. In addition, Section 5 chapters discuss RNAi utilization in various immune and infectious diseases. Section 6 chapters present the latest advances of RNAi application in studies of insects and parasitic pests such as ticks. All of these chapters highlight various aspects of RNAi and add interesting insights to the present RNAi discussions.
6. Safety and risk assessment
Manipulation of the organisms’ own genetic sequence signature(s) (cis-genesis) is usually considered safer compared to “trans-genesis” that utilizes “foreign” genetic material to create genetically modified (GM) crops and its products . However, for RNAi, when broken down to ~21 nucleotides this quickly may lose its meaning, as a trans-RNAi will only work if it has sufficient homology to an endogenous target transcript. Chemically, RNA is “
Safety concerns about RNAi-based drugs are exemplified by the lethality of 23 out of 49 distinct RNAi therapy experiments in mice because of potential "off-target" effects that could shut down non-targeted gene(s) with sequence similarity to therapeutic RNAi inducer . This observed lethality, however, could be due to “oversaturation" of the dsRNA pathway and delivery issues of short hairpin RNAs  that needs to be optimized for harmless therapeutic applications. There are several suggested approaches to minimize or eliminate such “off-target”, “oversaturation” or delivery issues, in particular through the use of (1) comprehensive
There may also be concerns about the uptake of intact plant miRNA by consumers through plant diet. Plant microRNAs and some long dsRNA molecules, with sequence complimentary and perfect matches to endogenous human genes, were demonstrated to survive the digestive tract of humans and can freely and routinely enter the blood system [67, 81].
Risk assessment and available protocols/guidelines are in the early stages of development. Some suggest that dsRNA-derived products must be subject to risk assessment studies . Other findings indirectly support the safety of RNAi [81, 82], provided its use is within specific dosage ranges, the correct delivery system is in place and RNAi inducers without possible off-target effects, unintended gene silencing and secondary dsRNA production can be designed. However, it is always advisable to admit to possible risks of any novel genomic technology, including RNAi, and consider potential biohazards and evaluate risks for environmental health, before release of a new product [58, 81–85]. To accomplish this, Heinemann et al.  proposed the following five-step guidance: (1) to perform detailed
7. Conclusions and future perspectives
Thus, being a revolutionizing discovery in genome biology to characterize functions of any desired unknown genetic sequences, the discovery of RNAi has significantly widened our knowledge on core cellular processes. This knowledge has created opportunities and solutions to longstanding obstacles in conventional agriculture and medicine, offering a bright future to curing complex human and animal diseases, improve crop production and protection, and a sustained global food security through proper manipulation of key genes with agricultural or medicinal importance. Although key issues on specificity, selectivity, and delivery of RNAi inducing structures still exist, and some safety risks associated with the use of RNAi products have been recognized, the general believe is that RNAi is a safer technology than trans-genomics utilizing “foreign” genetic information. Safe applications, however, require proper designing, dosage and delivery of RNAi inducers, and before its delivery for wide consumer market, the safety risks should be assessed. Addressing the advances made over the past three decades in RNAi research and commercialization, in this book, we have compiled and presented a diverse collection of chapters contributed by the science research communities. We all believe that RNAi, in combination with the rapidly expanding genomic information in key organisms and novel genome editing tools, will become even more powerful and efficient, and that we will all enjoy its benefits far into the future.
I thank the Academy of Sciences of Uzbekistan and Committee for Coordination Science and Technology Development of Uzbekistan for basic science FA-F5-T030; and several applied (FA-A6-T081 and FA-A6-T085) and innovation (I-2015-6-15/2 and I5-FQ-0-89-870) research grants. I am particularly grateful and thank the Office of International Research Programs (OIRP) of the United States Department of Agriculture (USDA) – Agricultural Research Service (ARS) and U.S. Civilian Research & Development Foundation (CRDF) for international cooperative grants P121, P121B, and UZB-TA-2292, which are devoted to study, development, application, risk assessment, and commercialization of RNAi cotton cultivars and their products. I sincerely thank Dr. Alexander R. van der Krol, Waginengen University, Netherlands, and Dr. Eric J. Devor, Iowa State University, the USA, for their critical reading and suggestions to this chapter manuscript.
Hannon GJ. RNA interference. Nature. 2002;418:244–51. DOI: 10.1038/418244a
Rothstein SJ, Dimaio J, Strand M, Rice D. Stable and heritable inhibition of the expression of nopaline synthase in tobacco expressing antisense RNA. Proc Natl Acad Sci U S A. 1987;84:8439–8443. DOI:10.1073/pnas.84.23.8439
van der Krol AR, Lenting PE, Veenstra J, van der Meer IM, Koes RE, Gerats AGM, Joseph N. M. Mol JNM, Stuitje AR. An anti-sense chalcone synthase gene in transgenic plants inhibits flower pigmentation. Nature 1988;333:866–869.DOI:10.1038/333866a0
van der Krol AR, Mur LA, Beld M, Mol JN, Stuitje AR. Flavonoid genes in petunia: addition of a limited number of gene copies may lead to a suppression of gene expression. Plant Cell. 1990;2:291-–99. DOI:10.1105/tpc.2.4.291
Napoli C, Lemieux C, Jorgensen R. Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans". Plant Cell. 1990; 2:279–289. DOI:10.1105/tpc.2.4.279
van Blokland R, van der Geest N, Mol JNM, Kooter JM. Transgene-mediated suppression of chalcone synthase expression in Petunia hybrida results from an increase in RNA turnover. Plant J. 1994;6:861–877. DOI:10.1046/j.1365-313X.1994.6060861.x.
Mol JNM, van der Krol AR. Antisense nucleic acids and proteins: fundamentals and applications. New York: Marcel Dekker; 1991. 231 p. ISBN 0-8247-8516-9.
Romano N, Macino G (1992). Quelling: transient inactivation of gene expression in Neurospora crassaby transformation with homologous sequences. Mol Microbiol. 1992;6:3343–3353. DOI:10.1111/j.1365-2958.1992.tb02202.x
Guo S, Kemphues K. par-1, a gene required for establishing polarity in C. elegansembryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell. 1995;81:611–620. DOI:10.1016/0092-8674(95)90082-9
Pal-Bhadra M, Bhadra U, Birchler J. Cosuppression in Drosophila: gene silencing of alcohol dehydrogenase by white-Adh transgenes is polycomb dependent. Cell. 1997;90: 479–490. DOI:10.1016/S0092-8674(00)80508-5
Fire A, Xu S, Montgomery M, Kostas S, Driver S, Mello C. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998;391: 806–811. DOI:10.1038/35888. PMID 9486653
Ratcliff F, Harrison B, Baulcombe D. A similarity between viral defense and gene silencing in plants. Science. 1997;276:1558. DOI:10.1126/science.276.5318.1558.
Godge MR, Purkayastha A, Dasgupta I, Kumar PP.Virus-induced gene silencing for functional analysis of selected genes. Plant Cell Rep. 2008;27: 209-219. DOI:
Hale C, Kleppe K, Terns RM, Terns MP. Prokaryotic silencing (psi)RNAs in Pyrococcus furiosus. RNA. 2008;14:2572–2579. DOI: 10.1261/rna.1246808
Abdurakhmonov IY, Buriev ZT, Saha S, Jenkins JN, Abdukarimov A, Pepper AE. Cotton PHYA1RNAi enhances major fiber quality and agronomic traits of cotton (Gossypium hirsutum L). Nature Communications.2014;4:3062; DOI:10. 1038/ncomms4062
Kupferschmidt K. A Lethal Dose of RNA. Science. 2013;341:732–733. DOI:10.1126/science.341.6147.732
Google Scholar. 2015. Available from: http://scholar.google.com [Accessed 2015-09-06]
PubMed database. 2015. Available from: http://www.ncbi.nlm.nih.gov/pubmed [Accessed from 2015-09-06]
Matranga C, Tomari Y, Shin C, Bartel D, Zamore P. Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes. Cell.2005;123: 607–620. DOI:10.1016/j.cell.2005.08.044
Leuschner P, Ameres S, Kueng S, Martinez J. Cleavage of the siRNA passenger strand during RISC assembly in human cells. EMBO Rep. 2006;7:314–320. DOI:10.1038/sj.embor.7400637
Haley B, Zamore B. Kinetic analysis of the RNAi enzyme complex. Nature Structural & Molecular Biology. 2004;11:599–606. DOI:10.1038/nsmb780
Tomari Y, Matranga C, Haley B, Martinez N, Zamore P; Matranga; Haley; Martinez; Zamore. A protein sensor for siRNA asymmetry. Science. 2004;306:1377–1380. DOI:10.1126/science.1102755
Sen G, Blau H. Argonaute 2/RISC resides in sites of mammalian mRNA decay known as cytoplasmic bodies. Nat Cell Biol. 2005;7: 633–636. DOI:10.1038/ncb1265. PMID 15908945
Lian S, Jakymiw A, Eystathioy T, Hamel J, Fritzler M, Chan E. GW bodies, microRNAs and the cell cycle. Cell Cycle. 2006;5:242–245. DOI:10.4161/cc.5.3.2410
Jakymiw A, Lian S, Eystathioy T, Li S, Satoh M, Hamel J, Fritzler M, Chan E. Disruption of P bodies impairs mammalian RNA interference. Nat Cell Biol.2005;7:1267–1274. DOI:10.1038/ncb1334
Parker G, Eckert D, Bass B. RDE-4 preferentially binds long dsRNA and its dimerization is necessary for cleavage of dsRNA to siRNA. RNA. 2006;12:807–818. DOI:10.1261/rna.2338706
Liu Q, Rand T, Kalidas S, Du F, Kim H, Smith D, Wang X. R2D2, a bridge between the initiation and effector steps of the Drosophila RNAi pathway. Science. 2003;301: 1921–1925. DOI:10.1126/science.1088710
Lim LP, Lau NC, Garrett-Engele P, Grimson A, Schelter JM, Castle J, Bartel DP, Linsley PS, Johnson JM. Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature. 2005;433:769–773. DOI:10.1038/nature03315. PMID 15685193
Selbach M, Schwanhäusser B, Thierfelder N, Fang Z, Khanin R, Rajewsky N. Widespread changes in protein synthesis induced by microRNAs. Nature. 2008:455: 58–63. DOI:10.1038/nature07228
Baek D, Villén J, Shin C, Camargo FD, Gygi SP, Bartel DP.The impact of microRNAs on protein output. Nature.2008;455: 64–71. DOI:10.1038/nature07242
Pillai RS, Bhattacharyya SN, Filipowicz W. Repression of protein synthesis by miRNAs: how many mechanisms? Trends Cell Biol 2007;17:118–126. DOI:10.1016/j.tcb.2006.12.007
Okamura K, Ishizuka A, Siomi H, Siomi M (2004). "Distinct roles for Argonaute proteins in small RNA-directed RNA cleavage pathways". Genes Dev. 2004;18:1655–1666. DOI:10.1101/gad.1210204
Lee Y, Nakahara K, Pham J, Kim K, He Z, Sontheimer E, Carthew R. Distinct roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways. Cell. 2004;117:69–81. DOI:10.1016/S0092-8674(04)00261-2
Vasudevan S, Tong Y, Steitz JA. Switching from Repression to Activation: MicroRNAs Can Up-Regulate Translation. Science. 2007;318:1931–1934. DOI:10.1126/science
Holmquist G, Ashley T. Chromosome organization and chromatin modification: influence on genome function and evolution. Cytogenet Genome Res.2006;114:96–125. DOI:10.1159/000093326
Verdel A, Jia S, Gerber S, Sugiyama T, Gygi S, Grewal S, Moazed D. RNAi-mediated targeting of heterochromatin by the RITS complex. Science. 2004;303:672–676. DOI:10.1126/science.1093686
Li LC, Okino ST, Zhao H, Pookot D, Place RF, Urakami S, Enokida H, Dahiya R. Small dsRNAs induce transcriptional activation in human cells. Proc Natl Acad Sci USA.2006;103:17337–17342. DOI:10.1073/pnas.0607015103
Check E. RNA interference: hitting the on switch. Nature. 2007;448: 855–858.DOI:10.1038/448855a
Stram Y, Kuzntzova L. Inhibition of viruses by RNA interference. Virus Genes. 2006;32:299–306. DOI:10.1007/s11262-005-6914-0
Lodish H, Berk A, Matsudaira P, Kaiser CA, Krieger M, Scott MP, Zipurksy SL, Darnell J. Molecular Cell Biology. 5th ed. New York; W.H; 2004.963 p. ISBN 978-0-7167-4366-8.
Jones L, Ratcliff F, Baulcombe DC. RNA-directed transcriptional gene silencing in plants can be inherited independently of the RNA trigger and requires Met1 for maintenance. Curr Biol. 2001;11:747–757. DOI:10.1016/S0960-9822(01)00226-3
Saumet A, Lecellier CH. Anti-viral RNA silencing: do we look like plants ?. Retrovirology. 2006;3:3. DOI:10.1186/1742-4690-3-3
DaRocha W, Otsu K, Teixeira S, Donelson J. Tests of cytoplasmic RNA interference (RNAi) and construction of a tetracycline-inducible T7 promoter system in Trypanosoma cruzi. Mol Biochem Parasitol. 2004;133:175–86. DOI:10.1016/j.molbiopara.2003.10.005
Robinson K, Beverley S. Improvements in transfection efficiency and tests of RNA interference (RNAi) approaches in the protozoan parasite Leishmania. Mol Biochem Parasitol. 2003;28:217–228. DOI:10.1016/S0166-6851(03)00079-3
Aravind L, Watanabe H, Lipman DJ, Koonin EJ. Lineage-specific loss and divergence of functionally linked genes in eukaryotes. Proc Natl Acad Sci USA. 2000;97:11319–11324. DOI:10.1073/pnas.200346997
Drinnenberg IA, Weinberg DE, Xie KT, Nower JP, Wolfe KH, Fink GR, Bartel DP. RNAi in budding yeast. Science 2009;326:544–550. DOI:10.1126/science.
Morita T, Mochizuki Y, Aiba H. Translational repression is sufficient for gene silencing by bacterial small noncoding RNAs in the absence of mRNA destruction". Proc Natl Acad Sci USA. 2006;103:4858–486. DOI:10.1073/pnas.0509638103
Larson MH, Gilbert LA, Wang X, Lim WA, Weissman JS, Qi LS. CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nature Protocols 2013;8:2180–2196. DOI:10.1038/nprot.2013.132
Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE, Stern-Ginossar N, Brandman O, Whitehead EH, Doudna JA, Lim WA, Weissman JS, Qi LS. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell. 2013;154:442–451. DOI:10.1016/j.cell.2013.06.044
Cerutti H, Casas-Mollano J. On the origin and functions of RNA-mediated silencing: from protists to man". Curr Genet. 2006;50:81–99. DOI:10.1007/s00294-006-0078-x
Buchon N, Vaury C. RNAi: a defensive RNA-silencing against viruses and transposable elements. Heredity. 2006;96:195–202. DOI:10.1038/sj.hdy.6800789.
Shabalina S, Koonin EV. Origins and evolution of eukaryotic RNA interference Ecol Evol. 2008;23:578–587. DOI: 10.1016/j.tree.2008.06.005
Anantharaman V, Koonin E, Aravind L. Comparative genomics and evolution of proteins involved in RNA metabolism. Nucleic Acids Res. 2002;30:1427–64. DOI:10.1093/nar/30.7.1427
Obbard DJ; Jiggins FM; Halligan DL; Little TJ. Natural selection drives extremely rapid evolution in antiviral RNAi genes. Curr Biol. 2006;16:580–585. DOI:10.1016/j.cub.2006.01.065
Lucy A, Guo H, Li W, Ding S. Suppression of post-transcriptional gene silencing by a plant viral protein localized in the nucleus. EMBO J. 2000;19:1672–1680. DOI:10.1093/emboj/19.7.1672
Hu Y, Stenlid J, Elfstrand M, Olson A. Evolution of RNA interference proteins dicer and argonaute in Basidiomycota. Mycologia. 2013;105:1489–1498.DOI: 10.3852/13-171
Voorhoeve PM, Agami R. Knockdown stands up. Trends Biotechnol. 2003;21:2–4. DOI:10.1016/S0167-7799(02)00002-1
Saurabh Satyajit, Vidyarthi AS, Prasad D. RNA interference: concept to reality in crop improvement. Planta. 2014;239:543–564. DOI:10.1007/s00425-013-2019-5
Sunilkumar G, Campbell L, Puckhaber L; Stipanovic R, Rathore K. Engineering cottonseed for use in human nutrition by tissue-specific reduction of toxic gossypol. Proc Natl Acad Sci USA.2006:103:18054–18059. DOI:10.1073/pnas.0605389103
Siritunga D, Sayre R. Generation of cyanogen-free transgenic cassava. Planta. 2003;217:367–373. DOI:10.1007/s00425-003-1005-8
Le L, Lorenz Y, Scheurer, S, Fötisch K, Enrique E, Bartra J, Biemelt S, Vieths S, Sonnewald U. Design of tomato fruits with reduced allergenicity by dsRNAi-mediated inhibition of ns-LTP (Lyc e 3) expression. Plant Biotechnol J. 2006;4:231–242. DOI:10.1111/j.1467-7652.2005.00175.x
Niggeweg R, Michael A, Martin C.Engineering plants with increased levels of the antioxidant chlorogenic acid. Nat Biotechnol.2004;22:746–54. DOI:10.1038/nbt966
Gavilano L, Coleman N, Burnley L, Bowman M, Kalengamaliro, Hayes A, Bush L, Siminszky B. Genetic engineering of Nicotiana tabacum for reduced nornicotine content". J Agric Food Chem. 2006;54:9071–9078. DOI:10.1021/jf0610458
Allen R, Millgate A, Chitty J, Thisleton J, Miller J, Fist A, Gerlach W, Larkin P. RNAi-mediated replacement of morphine with the nonnarcotic alkaloid reticuline in opium poppy". Nat Biotechnol.2004;22:1559–1566. DOI:10.1038/nbt1033
Zadeh A, Foster G. Transgenic resistance to tobacco ringspot virus. Acta Virol.2004;48:145–152
Mao YB, Cai WJ, Wang JW, Hong GJ, Tao XY, Wang LJ, Huang YP, Chen XY. Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol. Nat Biotechnol. 2007;25:1307–1313. DOI: 10.1038/nbt1352
Heinemann JA, Agapito-Tenfen SZ, Carman JA. A comparative evaluation of the regulation of GM crops or products containing dsRNA and suggested improvements to risk assessments. Environ Int. 2013;55:43–55. DOI: 10.1016/j.envint.2013.02.010
Crowe S. Suppression of chemokine receptor expression by RNA interference allows for inhibition of HIV-1 replication. AIDS. 2003;17 Suppl 4:S103–S105. DOI:10.1097/00002030-200317004-00014
Kusov Y, Kanda T, Palmenberg A, Sgro J, Gauss-Müller V. Silencing of Hepatitis A Virus Infection by Small Interfering RNAs. J Virol. 2006;80:5599–610. DOI:10.1128/JVI.01773-05
Jia F, Zhang Y, Liu C.A retrovirus-based system to stably silence hepatitis B virus genes by RNA interference. Biotechnol Lett. 2006;8:1679–1685. DOI:10.1007/s10529-006-9138-z
Hu L, Wang Z, Hu C, Liu X, Yao L, Li W, Qi Y. Inhibition of Measles virus multiplication in cell culture by RNA interference. Acta Virol. 2005;49:227–234. DOI: Not availble
Putral L, Gu W, McMillan N. RNA interference for the treatment of cancer". Drug News Perspect. 2006;19:317–324. DOI:10.1358/dnp.2006.19.6.985937
Izquierdo M. Short interfering RNAs as a tool for cancer gene therapy". Cancer Gene Ther. 2005;12:217–227. DOI:10.1038/sj.cgt.7700791
Li C, Parker A, Menocal E, Xiang S, Borodyansky L, Fruehauf J. Delivery of RNA interference. Cell Cycle. 2006;5:2103–2109. DOI:10.4161/cc.5.18.3192
Takeshita F, Ochiya T. Therapeutic potential of RNA interference against cancer. Cancer Sci. 2006;97:689–696. DOI:10.1111/j.1349-7006.2006.00234.x
Raoul C, Barker S, Aebischer P. Viral-based modelling and correction of neurodegenerative diseases by RNA interference. Gene Ther. 2006;3:487–495. DOI:10.1038/sj.gt.3302690
Hou H, Atlihan N, Lu ZX. New biotechnology enhances the application of cisgenesis in plant breeding. Front Plant Sci. 2014;5:389. DOI: 10.3389/fpls.2014.00389.
Check E. RNA treatment kills mice. Nature. 2006; DOI:10.1038/news060522-10
Grimm D, Streetz K, Jopling C, Storm T; Pandey K, Davis C, Marion P, Salazar Kay F, Kay M. Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Nature. 2006;441:537–541. DOI:10.1038/nature04791
Qiu S, Adema C, Lane T. A computational study of off-target effects of RNA interference. Nucleic Acids Res. 2005;33:1834–1847. DOI:10.1093/nar/gki324
Petrick JS, Brower-Toland B, Jackson AL, Kier LD. Safety assessment of food and feed from biotechnology-derived crops employing RNA-mediated gene regulation to achieve desired traits: a scientific review. Regul Toxicol Pharmacol. 2013;66:167–176. DOI: 10.1016/j.yrtph.2013.03.008
Kamthan A, Chaudhuri A, Kamthan M, Datta A. Small RNAs in plants: recent development and application for crop improvement. Front Plant Sci. 2015;6:208. DOI: 10.3389/fpls.2015.00208
Lemgo GN, Sabbadini S, Pandolfini T, Mezzetti B. Biosafety considerations of RNAi-mediated virus resistance in fruit-tree cultivars and in rootstock. Transgenic Res. 2013;22:1073–1088. DOI: 10.1007/s11248-013-9728-1.
Ramesh SV. Non-coding RNAs in crop genetic modification: considerations and predictable environmental risk assessments (ERA). Mol Biotechnol. 2013;55:87–100. DOI: 10.1007/s12033-013-9648-6
Auer C, Frederick R. Crop improvement using small RNAs: applications and predictive ecological risk assessments. Trends Biotechnol. 2009;27:644–651. DOI: 10.1016/j.tibtech.2009.08.005