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Methods in Molecular Biology (Clifton,... 2018RNA interference (RNAi) is a biological process by which double-stranded RNA (dsRNA) induces sequence-specific gene silencing by targeting mRNA for degradation. As a... (Review)
Review
RNA interference (RNAi) is a biological process by which double-stranded RNA (dsRNA) induces sequence-specific gene silencing by targeting mRNA for degradation. As a tool for knocking down the expression of individual genes posttranscriptionally, RNAi has been widely used to study the cellular function of genes. In this chapter, I describe procedures for using gene-specific, synthetic, short interfering RNA (siRNA) to induce gene silencing in mammalian cells. Protocols for using lipid-based transfection reagents and electroporation techniques are provided. Potential challenges and problems associated with the siRNA technology are also discussed.
Topics: Animals; Electroporation; Gene Knockdown Techniques; Humans; RNA Interference; RNA Stability; Transfection
PubMed: 29423805
DOI: 10.1007/978-1-4939-7471-9_16 -
Expert Opinion on Therapeutic Targets Apr 2020: Amyotrophic lateral sclerosis (ALS) is a progressive and incurable neurodegenerative disorder that targets upper and lower motor neurons and leads to fatal muscle... (Review)
Review
: Amyotrophic lateral sclerosis (ALS) is a progressive and incurable neurodegenerative disorder that targets upper and lower motor neurons and leads to fatal muscle paralysis. Mutations in the superoxide dismutase 1 (SOD1) gene are responsible for 15% of familial ALS cases, but several studies have indicated that SOD1 dysfunction may also play a pathogenic role in sporadic ALS. SOD1 induces numerous toxic effects through the pathological misfolding and aggregation of mutant SOD1 species, hence a reduction of the levels of toxic variants appears to be a promising therapeutic strategy for SOD1-related ALS. Several methods are used to modulate gene expression ; these include RNA interference, antisense oligonucleotides (ASOs) and CRISPR/Cas9 technology.: This paper examines the current approaches for gene silencing and the progress made in silencing SOD1 . It progresses to shed light on the key results and pitfalls of these studies and highlights the future challenges and new perspectives for this exciting research field.: Gene silencing strategies targeting SOD1 may represent effective approaches for familial and sporadic ALS-related neurodegeneration; however, the risk of off-target effects must be minimized, and effective and minimally invasive delivery strategies should be fine-tuned.
Topics: Amyotrophic Lateral Sclerosis; Animals; CRISPR-Cas Systems; Gene Expression Regulation; Gene Silencing; Humans; Molecular Targeted Therapy; Mutation; Oligonucleotides, Antisense; RNA Interference; Superoxide Dismutase-1
PubMed: 32125907
DOI: 10.1080/14728222.2020.1738390 -
FEBS Letters Oct 2005RNA interference (RNAi) is a form of gene silencing induced by double stranded RNA (dsRNA) that is processed into short interfering RNAs (siRNAs). RNAi can induce both... (Review)
Review
RNA interference (RNAi) is a form of gene silencing induced by double stranded RNA (dsRNA) that is processed into short interfering RNAs (siRNAs). RNAi can induce both post-transcriptional and transcriptional gene silencing. In Caenorhabditis elegans, there are several distinct pathways where post-transcriptional or/and transcriptional RNAi mechanisms are involved. RNAi in C. elegans is also systemic and heritable. This review will discuss RNAi related pathways, features of RNAi in C. elegans and possibilities of endogenous gene regulation by RNAi.
Topics: Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Gene Silencing; Models, Biological; Models, Genetic; Phenotype; RNA Interference; RNA Processing, Post-Transcriptional; RNA, Small Interfering; Transcription, Genetic
PubMed: 16162338
DOI: 10.1016/j.febslet.2005.08.001 -
Journal of Genetics Dec 2015X-chromosome inactivation, which was discovered by Mary Lyon in 1961 results in random silencing of one X chromosome in female mammals. This review is dedicated to Mary... (Review)
Review
X-chromosome inactivation, which was discovered by Mary Lyon in 1961 results in random silencing of one X chromosome in female mammals. This review is dedicated to Mary Lyon, who passed away last year. She predicted many of the features of X inactivation, for e.g., the existence of an X inactivation center, the role of L1 elements in spreading of silencing and the existence of genes that escape X inactivation. Starting from her published work here we summarize advances in the field.
Topics: Animals; Gene Silencing; Humans; X Chromosome; X Chromosome Inactivation
PubMed: 26690513
DOI: 10.1007/s12041-015-0574-1 -
Archives of Disease in Childhood.... Aug 2016
Review
Topics: Adolescent; CRISPR-Associated Proteins; CRISPR-Cas Systems; Child; Child, Preschool; Clustered Regularly Interspaced Short Palindromic Repeats; Disease Management; Female; Gene Silencing; Humans; Infant; Infant, Newborn; Male; Pediatrics; Young Adult
PubMed: 27059283
DOI: 10.1136/archdischild-2016-310459 -
Genes & Development Jun 2020The X inactive-specific transcript () gene is the master regulator of X chromosome inactivation in mammals. produces a long noncoding (lnc)RNA that accumulates over the... (Review)
Review
The X inactive-specific transcript () gene is the master regulator of X chromosome inactivation in mammals. produces a long noncoding (lnc)RNA that accumulates over the entire length of the chromosome from which it is transcribed, recruiting factors to modify underlying chromatin and silence X-linked genes in Recent years have seen significant progress in identifying important functional elements in Xist RNA, their associated RNA-binding proteins (RBPs), and the downstream pathways for chromatin modification and gene silencing. In this review, we summarize progress in understanding both how these pathways function in Xist-mediated silencing and the complex interplay between them.
Topics: DNA-Binding Proteins; Gene Silencing; Methyltransferases; Proteins; RNA, Long Noncoding; RNA-Binding Proteins; Receptors, Cytoplasmic and Nuclear; X Chromosome Inactivation; Lamin B Receptor
PubMed: 32482714
DOI: 10.1101/gad.337196.120 -
Trends in Biochemical Sciences Oct 2021Transfer RNA (tRNA)-derived small RNAs (tsRNAs) are among the most ancient small RNAs in all domains of life and are generated by the cleavage of tRNAs. Emerging studies... (Review)
Review
Transfer RNA (tRNA)-derived small RNAs (tsRNAs) are among the most ancient small RNAs in all domains of life and are generated by the cleavage of tRNAs. Emerging studies have begun to reveal the versatile roles of tsRNAs in fundamental biological processes, including gene silencing, ribosome biogenesis, retrotransposition, and epigenetic inheritance, which are rooted in tsRNA sequence conservation, RNA modifications, and protein-binding abilities. We summarize the mechanisms of tsRNA biogenesis and the impact of RNA modifications, and propose how thinking of tsRNA functionality from an evolutionary perspective urges the expansion of tsRNA research into a wider spectrum, including cross-tissue/cross-species regulation and harnessing of the 'tsRNA code' for precision medicine.
Topics: Gene Silencing; RNA, Transfer
PubMed: 34053843
DOI: 10.1016/j.tibs.2021.05.001 -
Cell Research Jun 2021RNAi therapy has undergone two stages of development, direct injection of synthetic siRNAs and delivery with artificial vehicles or conjugated ligands; both have not...
RNAi therapy has undergone two stages of development, direct injection of synthetic siRNAs and delivery with artificial vehicles or conjugated ligands; both have not solved the problem of efficient in vivo siRNA delivery. Here, we present a proof-of-principle strategy that reprogrammes host liver with genetic circuits to direct the synthesis and self-assembly of siRNAs into secretory exosomes and facilitate the in vivo delivery of siRNAs through circulating exosomes. By combination of different genetic circuit modules, in vivo assembled siRNAs are systematically distributed to multiple tissues or targeted to specific tissues (e.g., brain), inducing potent target gene silencing in these tissues. The therapeutic value of our strategy is demonstrated by programmed silencing of critical targets associated with various diseases, including EGFR/KRAS in lung cancer, EGFR/TNC in glioblastoma and PTP1B in obesity. Overall, our strategy represents a next generation RNAi therapeutics, which makes RNAi therapy feasible.
Topics: Gene Silencing; Glioblastoma; Humans; RNA Interference; RNA, Small Interfering; RNAi Therapeutics
PubMed: 33782530
DOI: 10.1038/s41422-021-00491-z -
Nature Protocols Nov 2013Sequence-specific control of gene expression on a genome-wide scale is an important approach for understanding gene functions and for engineering genetic regulatory...
Sequence-specific control of gene expression on a genome-wide scale is an important approach for understanding gene functions and for engineering genetic regulatory systems. We have recently described an RNA-based method, CRISPR interference (CRISPRi), for targeted silencing of transcription in bacteria and human cells. The CRISPRi system is derived from the Streptococcus pyogenes CRISPR (clustered regularly interspaced palindromic repeats) pathway, requiring only the coexpression of a catalytically inactive Cas9 protein and a customizable single guide RNA (sgRNA). The Cas9-sgRNA complex binds to DNA elements complementary to the sgRNA and causes a steric block that halts transcript elongation by RNA polymerase, resulting in the repression of the target gene. Here we provide a protocol for the design, construction and expression of customized sgRNAs for transcriptional repression of any gene of interest. We also provide details for testing the repression activity of CRISPRi using quantitative fluorescence assays and native elongating transcript sequencing. CRISPRi provides a simplified approach for rapid gene repression within 1-2 weeks. The method can also be adapted for high-throughput interrogation of genome-wide gene functions and genetic interactions, thus providing a complementary approach to RNA interference, which can be used in a wider variety of organisms.
Topics: Bacterial Proteins; Clustered Regularly Interspaced Short Palindromic Repeats; Gene Expression Regulation; Gene Silencing; Genetic Techniques; RNA Interference; Streptococcus pyogenes
PubMed: 24136345
DOI: 10.1038/nprot.2013.132 -
Journal of Visualized Experiments : JoVE Jun 2009RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA. This silencing mechanism uses two major classes of RNA regulators: microRNAs,...
RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host. Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript. Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa.
Topics: Gene Silencing; Solanum lycopersicum; Plant Viruses; RNA Interference; Nicotiana
PubMed: 19516240
DOI: 10.3791/1292