Gene silencing or gene editing: the pros and cons.

Author(s):  
Huw D. Jones

Abstract Research into plant genetics often requires the suppression or complete knockout of gene expression to scientifically validate gene function. In addition, the phenotypes obtained from gene suppression can occasionally have commercial value for plant breeders. Until recently, the methodological choices to achieve these goals fell into two broad types: either some form of RNA-based gene silencing; or the screening of large numbers of natural or induced random genomic mutations. The more recent invention of gene editing as a tool for targeted mutation potentially gives researchers and plant breeders another route to block gene function. RNAi is widely used in animal and plant research and functions to silence gene expression by degrading the target gene transcript. Although RNAi offers unique advantages over genomic mutations, it often leads to the formation of a genetically modified organism (GMO), which for commercial activities has major regulatory and acceptance issues in some regions of the world. Traditional methods of generating genomic mutations are more laborious and uncertain to achieve the desired goals but possess a distinct advantage of not being governed by GMO regulations. Gene editing (GE) technologies have some of the advantages of both RNAi and classical mutation breeding in that they can be designed to give simple knockouts or to modulate gene expression more subtly. GE also has a more complex regulatory position, with some countries treating it as another conventional breeding method whilst the EU defines GE as a technique of genetic modification and applies the normal GMO authorization procedures. This chapter explores the pros and cons of RNAi alongside other methods of modulating gene function.

Author(s):  
Huw D. Jones

Abstract Research into plant genetics often requires the suppression or complete knockout of gene expression to scientifically validate gene function. In addition, the phenotypes obtained from gene suppression can occasionally have commercial value for plant breeders. Until recently, the methodological choices to achieve these goals fell into two broad types: either some form of RNA-based gene silencing; or the screening of large numbers of natural or induced random genomic mutations. The more recent invention of gene editing as a tool for targeted mutation potentially gives researchers and plant breeders another route to block gene function. RNAi is widely used in animal and plant research and functions to silence gene expression by degrading the target gene transcript. Although RNAi offers unique advantages over genomic mutations, it often leads to the formation of a genetically modified organism (GMO), which for commercial activities has major regulatory and acceptance issues in some regions of the world. Traditional methods of generating genomic mutations are more laborious and uncertain to achieve the desired goals but possess a distinct advantage of not being governed by GMO regulations. Gene editing (GE) technologies have some of the advantages of both RNAi and classical mutation breeding in that they can be designed to give simple knockouts or to modulate gene expression more subtly. GE also has a more complex regulatory position, with some countries treating it as another conventional breeding method whilst the EU defines GE as a technique of genetic modification and applies the normal GMO authorization procedures. This chapter explores the pros and cons of RNAi alongside other methods of modulating gene function.


Development ◽  
1995 ◽  
Vol 121 (12) ◽  
pp. 3989-3996 ◽  
Author(s):  
J. Broadus ◽  
C.Q. Doe

In the Drosophila CNS, early neuroblast formation and fate are controlled by the pair-rule class of segmentation genes. The distantly related Schistocerca (grasshopper) embryo has a similar arrangement of neuroblasts, despite lack of known pair-rule gene function. Does divergent pair-rule gene function lead to different neuroblast identities, or can different patterning mechanisms produce homologous neuroblasts? We use four molecular markers to compare Drosophila and Schistocerca neuroblast identity: seven-up, prospero, engrailed, and fushi-tarazu/Dax. In both insects some early-forming neuroblasts share key features of neuroblast identity (position, time of formation, and temporally accurate gene expression); thus, different patterning mechanisms can generate similar neuroblast fates. In contrast, several later-forming neuroblasts show species-specific differences in position and/or gene expression; these neuroblast identities seem to have diverged, suggesting that evolution of the insect central nervous system can occur through changes in embryonic neuroblast identity.


Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 779
Author(s):  
Man Teng ◽  
Yongxiu Yao ◽  
Venugopal Nair ◽  
Jun Luo

In recent years, the CRISPR/Cas9-based gene-editing techniques have been well developed and applied widely in several aspects of research in the biological sciences, in many species, including humans, animals, plants, and even in viruses. Modification of the viral genome is crucial for revealing gene function, virus pathogenesis, gene therapy, genetic engineering, and vaccine development. Herein, we have provided a brief review of the different technologies for the modification of the viral genomes. Particularly, we have focused on the recently developed CRISPR/Cas9-based gene-editing system, detailing its origin, functional principles, and touching on its latest achievements in virology research and applications in vaccine development, especially in large DNA viruses of humans and animals. Future prospects of CRISPR/Cas9-based gene-editing technology in virology research, including the potential shortcomings, are also discussed.


Plant Methods ◽  
2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Decai Tuo ◽  
Peng Zhou ◽  
Pu Yan ◽  
Hongguang Cui ◽  
Yang Liu ◽  
...  

Abstract Background Cassava is an important crop for food security and industry in the least-developed and developing countries. The completion of the cassava genome sequence and identification of large numbers of candidate genes by next-generation sequencing provide extensive resources for cassava molecular breeding and increase the need for rapid and efficient gene function analysis systems in cassava. Several plant virus-induced gene silencing (VIGS) systems have been developed as reverse genetic tools for rapid gene function analysis in cassava. However, these VIGS vectors could cause severe viral symptoms or inefficient gene silencing. Results In this study, we constructed agroinfection-compatible infectious cDNA clones of cassava common mosaic virus isolate CM (CsCMV-CM, genus Potexvirus, family Alphaflexiviridae) that causes systemic infection with mild symptoms in cassava. CsCMV-CM was then modified to a viral vector carrying the Nimble cloning frame, which facilitates the rapid and high-throughput cloning of silencing fragments into the viral genome. The CsCMV-based vector successfully silenced phytoene desaturase (PDS) and magnesium chelatase subunit I (ChlI) in different cassava varieties and Nicotiana benthamiana. The silencing of the ChlI gene could persist for more than two months. Conclusions This CsCMV-based VIGS system provides a new tool for rapid and efficient gene function studies in cassava.


Molecules ◽  
2021 ◽  
Vol 26 (3) ◽  
pp. 701
Author(s):  
Tatiana S. Golubeva ◽  
Viktoria A. Cherenko ◽  
Konstantin E. Orishchenko

Selective regulation of gene expression by means of RNA interference has revolutionized molecular biology. This approach is not only used in fundamental studies on the roles of particular genes in the functioning of various organisms, but also possesses practical applications. A variety of methods are being developed based on gene silencing using dsRNA—for protecting agricultural plants from various pathogens, controlling insect reproduction, and therapeutic techniques related to the oncological disease treatment. One of the main problems in this research area is the successful delivery of exogenous dsRNA into cells, as this can be greatly affected by the localization or origin of tumor. This overview is dedicated to describing the latest advances in the development of various transport agents for the delivery of dsRNA fragments for gene silencing, with an emphasis on cancer treatment.


2013 ◽  
Vol 40 (10) ◽  
pp. 1029 ◽  
Author(s):  
Aguida M. A. P. Morales ◽  
Jamie A. O'Rourke ◽  
Martijn van de Mortel ◽  
Katherine T. Scheider ◽  
Timothy J. Bancroft ◽  
...  

Rpp4 (Resistance to Phakopsora pachyrhizi 4) confers resistance to Phakopsora pachyrhizi Sydow, the causal agent of Asian soybean rust (ASR). By combining expression profiling and virus induced gene silencing (VIGS), we are developing a genetic framework for Rpp4-mediated resistance. We measured gene expression in mock-inoculated and P. pachyrhizi-infected leaves of resistant soybean accession PI459025B (Rpp4) and the susceptible cultivar (Williams 82) across a 12-day time course. Unexpectedly, two biphasic responses were identified. In the incompatible reaction, genes induced at 12 h after infection (hai) were not differentially expressed at 24 hai, but were induced at 72 hai. In contrast, genes repressed at 12 hai were not differentially expressed from 24 to 144 hai, but were repressed 216 hai and later. To differentiate between basal and resistance-gene (R-gene) mediated defence responses, we compared gene expression in Rpp4-silenced and empty vector-treated PI459025B plants 14 days after infection (dai) with P. pachyrhizi. This identified genes, including transcription factors, whose differential expression is dependent upon Rpp4. To identify differentially expressed genes conserved across multiple P. pachyrhizi resistance pathways, Rpp4 expression datasets were compared with microarray data previously generated for Rpp2 and Rpp3-mediated defence responses. Fourteen transcription factors common to all resistant and susceptible responses were identified, as well as fourteen transcription factors unique to R-gene-mediated resistance responses. These genes are targets for future P. pachyrhizi resistance research.


BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Shuhua Zhan ◽  
Cortland Griswold ◽  
Lewis Lukens

Abstract Background Genetic variation for gene expression is a source of phenotypic variation for natural and agricultural species. The common approach to map and to quantify gene expression from genetically distinct individuals is to assign their RNA-seq reads to a single reference genome. However, RNA-seq reads from alleles dissimilar to this reference genome may fail to map correctly, causing transcript levels to be underestimated. Presently, the extent of this mapping problem is not clear, particularly in highly diverse species. We investigated if mapping bias occurred and if chromosomal features associated with mapping bias. Zea mays presents a model species to assess these questions, given it has genotypically distinct and well-studied genetic lines. Results In Zea mays, the inbred B73 genome is the standard reference genome and template for RNA-seq read assignments. In the absence of mapping bias, B73 and a second inbred line, Mo17, would each have an approximately equal number of regulatory alleles that increase gene expression. Remarkably, Mo17 had 2–4 times fewer such positively acting alleles than did B73 when RNA-seq reads were aligned to the B73 reference genome. Reciprocally, over one-half of the B73 alleles that increased gene expression were not detected when reads were aligned to the Mo17 genome template. Genes at dissimilar chromosomal ends were strongly affected by mapping bias, and genes at more similar pericentromeric regions were less affected. Biased transcript estimates were higher in untranslated regions and lower in splice junctions. Bias occurred across software and alignment parameters. Conclusions Mapping bias very strongly affects gene transcript abundance estimates in maize, and bias varies across chromosomal features. Individual genome or transcriptome templates are likely necessary for accurate transcript estimation across genetically variable individuals in maize and other species.


2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Mark E Pepin ◽  
David K Crossman ◽  
Joseph P Barchue ◽  
Salpy V Pamboukian ◽  
Steven M Pogwizd ◽  
...  

To identify the role of glucose in the development of diabetic cardiomyopathy, we had directly assessed glucose delivery to the intact heart on alterations of DNA methylation and gene expression using both an inducible heart-specific transgene (glucose transporter 4; mG4H) and streptozotocin-induced diabetes (STZ) mouse models. We aimed to determine whether long-lasting diabetic complications arise from prior transient exposure to hyperglycemia via a process termed “glycemic memory.” We had identified DNA methylation changes associated with significant gene expression regulation. Comparing our results from STZ, mG4H, and the modifications which persist following transgene silencing, we now provide evidence for cardiac DNA methylation as a persistent epigenetic mark contributing to glycemic memory. To begin to determine which changes contribute to human heart failure, we measured both RNA transcript levels and whole-genome DNA methylation in heart failure biopsy samples (n = 12) from male patients collected at left ventricular assist device placement using RNA-sequencing and Methylation450 assay, respectively. We hypothesized that epigenetic changes such as DNA methylation distinguish between heart failure etiologies. Our findings demonstrated that type 2 diabetic heart failure patients (n = 6) had an overall signature of hypomethylation, whereas patients listed as ischemic (n = 5) had a distinct hypermethylation signature for regulated transcripts. The focus of this initial analysis was on promoter-associated CpG islands with inverse changes in gene transcript levels, from which diabetes (14 genes; e.g. IGFBP4) and ischemic (12 genes; e.g. PFKFB3) specific targets emerged with significant regulation of both measures. By combining our mouse and human molecular analyses, we provide evidence that diabetes mellitus governs direct regulation of cellular function by DNA methylation and the corresponding gene expression in diabetic mouse and human hearts. Importantly, many of the changes seen in either mouse type 1 diabetes or human type 2 diabetes were similar supporting a consistent mechanism of regulation. These studies are some of the first steps at defining mechanisms of epigenetic regulation in diabetic cardiomyopathy.


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