Gene Silencing – Definition, Process, Techniques, Applications

Gene silencing, a mechanism of reducing or suppressing gene expression, is critical in managing gene expression in cells. Genes, which are responsible for protein production, have distinct expression spectra that determine the amount of protein produced by a gene in a specific cell type. When the expression profile of genes is altered, however, it can result in a variety of health problems, including cancer. This is where gene silencing comes in.

In this essay, we will look at the significance of gene silencing, the various approaches used for it, and its numerous uses. Gene silence is the act of controlling gene expression to prevent specific genes from being expressed. It can occur during transcription or translation processes. While gene silencing approaches are routinely used in research, they are also increasingly being used in the creation of medicines to address diseases such as cancer, infectious diseases, and neurological disorders.

Although gene silencing and gene knockdown are typically used interchangeably, there is a differentiation between the two. Silenced genes have reduced expression, whereas knocked-out genes have been totally wiped from the organism’s DNA and have no expression. Methods for silencing genes, such as RNA interference (RNAi), CRISPR, or small interfering RNA (siRNA), often suppress gene expression by at least 70%, allowing researchers to study key genes required for animal survival. Because diseases are frequently connected with genes that have low expression, this technique provides a more comprehensive understanding of disease development.

In the following sections, we will explore more into gene silencing techniques and applications, emphasising its potential to advance our understanding of gene regulation and its therapeutic implications.

What is gene silencing? 

In modern genetic engineering research, gene silencing is a powerful gene-editing approach. It entails suppressing or controlling gene expression through the use of several approaches such as RNA interference, CRISPR-CAS9, and antisense RNA. Researchers can get insights into gene function and identify new therapeutic approaches by manipulating the expression of specific genes of interest.

Consider the case of the CASCADE gene to better appreciate the relevance of gene silence. This gene generates a protein that is essential for cell cycle regulation. However, in other situations, CASCADE activity becomes unbalanced, resulting in excessive protein synthesis.

CASCADE overactivity disturbs the regular cell cycle and promotes aberrant cell proliferation, both of which are hallmarks of cancer. To recover control over CASCADE expression, it is necessary to suppress or minimise its activity. This can be accomplished by inserting mutations into the CASCADE gene or by targeting the mRNA molecules it generates.

For example, scientists could use biomolecules that selectively target and degrade the mRNA of the CASCADE gene. By doing so, the related protein’s production is suppressed, allowing for precise control over its overexpression.

When contemplating the negative consequences of dysregulated gene expression, the motive for gene silencing becomes evident. Researchers can unravel the underlying causes of diseases and potentially develop novel remedies by changing gene expression.

It is worth mentioning that gene silence occurs naturally within cells as well as through artificial interventions. Cells have their own internal methods for controlling gene activity via gene silence, which helps to maintain appropriate gene expression and cellular homeostasis.

In the following sections, we will delve deeper into the various approaches used in gene silencing and investigate its wide applications in various sectors, emphasising its potential for scientific advancement and medicinal breakthroughs.

Definition of gene silencing

Gene silencing is a technique used in genetic engineering to quiet, repress, or limit the expression of specific genes or genes of interest.

Or

Gene silencing occurs when a cell suppresses gene activity to regulate its expression through a natural mechanism.

Importance of gene silencing

The significance of gene silencing extends beyond artificial procedures and includes natural processes that organisms use to survive. The CRISPR-CAS9 system found in bacteria, which serves as a defence mechanism against phages or viruses, is one famous example.

In this arrangement, the CAS9 nuclease attaches to the invading phage’s target nucleic acid and then destroys it. This defence mechanism enables bacteria to defend themselves against viral genes while preserving cellular integrity.

Gene silencing mechanisms exist in eukaryotes as well as prokaryotes, most notably through the activity of a certain type of non-coding RNA. These non-coding RNAs bind to certain mRNA transcripts and inactivate them, preventing them from being translated into functional proteins. In eukaryotes, this endogenous gene silencing mechanism is critical for controlling gene expression and maintaining cellular functions.

Researchers can investigate the enormous potential of gene silencing by harnessing these natural mechanisms and inventing artificial approaches. Specific genotypes linked with undesirable characteristics can be suppressed via artificial gene silencing approaches. This is especially useful in research and therapeutic applications since it allows for the study of gene functions and the creation of potential remedies.

Furthermore, gene silencing approaches can be employed to create new phenotypic combinations. Scientists can investigate the effects on biological processes and even produce unique features or characteristics by selectively silencing or manipulating individual genes.

The importance of gene silencing is derived from its ability to shed light on gene regulation, disease mechanisms, and prospective therapeutic approaches. Researchers can reveal the underlying causes of diseases, devise targeted treatments, and potentially open up new pathways for advancement in a variety of sectors by understanding and altering gene expression.

In the following sections, we will explore more into gene silencing methods and applications, emphasising their importance in scientific research, medicine, and biotechnology.

Types of gene silencing

1. Transcriptional Gene Silencing (TGS): Transcriptional gene silencing occurs at the level of gene transcription. It involves the inhibition of RNA polymerase from binding to the gene promoter region or the repression of transcriptional machinery. TGS can be mediated by various mechanisms such as DNA methylation, histone modification, and the action of transcriptional repressor proteins. These mechanisms lead to the compaction of chromatin structure, making the gene inaccessible for transcription factors and RNA polymerase.
2. Post-Transcriptional Gene Silencing (PTGS): Post-transcriptional gene silencing operates after the transcription of the gene has taken place. It involves the degradation or inhibition of the translation of messenger RNA (mRNA) molecules. PTGS is primarily mediated by small RNA molecules, including microRNAs (miRNAs) and small interfering RNAs (siRNAs). These small RNA molecules bind to complementary sequences on the target mRNA, leading to its degradation or blocking its translation into a protein.
3. RNA Interference (RNAi): RNA interference is a specific type of gene silencing that is triggered by the introduction of double-stranded RNA (dsRNA) molecules into cells. The dsRNA molecules are processed into siRNAs by an enzyme called Dicer. These siRNAs then guide a protein complex called RNA-induced silencing complex (RISC) to target and degrade the complementary mRNA molecules, resulting in gene silencing. RNAi is a powerful tool used in research and has potential therapeutic applications.
4. Epigenetic Gene Silencing: Epigenetic modifications refer to heritable changes in gene expression that do not involve alterations in the underlying DNA sequence. Epigenetic gene silencing occurs through mechanisms such as DNA methylation and histone modifications. DNA methylation involves the addition of a methyl group to the DNA molecule, often resulting in the repression of gene expression. Histone modifications, including methylation, acetylation, and phosphorylation, can influence the accessibility of DNA to the transcriptional machinery.
5. Paramutation: Paramutation is a unique form of gene silencing observed in certain plants and animals. It involves the heritable modification of gene expression patterns between two alleles of the same gene, resulting in one allele being silenced or reduced in its expression. The silenced allele is then transmitted to subsequent generations, leading to stable and heritable gene silencing.
6. Transposons in Gene Silencing: Transposons, also known as jumping genes, are mobile genetic elements that can move around within a genome. In some cases, transposons can disrupt gene function by inserting themselves within a gene or its regulatory regions. This can lead to gene silencing, as the insertion of a transposon can interfere with proper gene expression. Transposons can also trigger the activation of gene silencing mechanisms, such as DNA methylation or histone modifications, to regulate their own mobility within the genome.
7. Antisense Oligonucleotides (ASOs): Antisense oligonucleotides are short synthetic DNA or RNA molecules designed to bind to specific mRNA sequences in a complementary manner. By binding to the target mRNA, ASOs can block its translation into a protein or induce the degradation of the mRNA molecule. This mechanism allows for specific gene silencing by preventing the production of a particular protein. ASOs have shown promise in therapeutic applications, especially in treating genetic disorders caused by the abnormal expression of specific genes.
8. CRISPR-Cas9 Gene Silencing: CRISPR-Cas9 is a revolutionary gene-editing tool that can also be used for gene silencing. CRISPR-Cas9 can be programmed to target specific DNA sequences within a genome. By guiding the Cas9 enzyme to a target gene, it can introduce double-stranded breaks in the DNA. This triggers DNA repair mechanisms that can lead to gene knockout or silencing. Additionally, a modified version of CRISPR-Cas9 called dCas9 can be used to target specific gene promoters and recruit repressor proteins or epigenetic modifiers, effectively silencing gene expression.

RNA interference-mediated gene silencing

• RNA interference (RNAi) is a naturally occurring cellular mechanism that regulates gene expression. Utilising small RNA molecules, specifically siRNA (small interfering RNA) and miRNA (microRNA), to silence or downregulate the expression of particular genes.
• Both siRNA and miRNA are noncoding RNAs, indicating that they do not code for proteins. Instead, they serve as regulatory molecules that control post-transcriptional gene expression. Typically composed of 20 to 30 nucleotides, these small RNA molecules have specific sequences that enable them to bind to complementary mRNA molecules.
• SiRNA functions by binding directly to the mRNA target, causing its degradation and consequent inhibition of protein synthesis. When introduced into a cell, siRNA binds to an enzyme known as RNA-induced silencing complex (RISC). The RISC unwinds the double-stranded siRNA molecule and directs the guide strand to the target mRNA. The guide strand binds to the mRNA’s complementary sequence, initiating a series of enzymatic reactions that ultimately lead to the mRNA’s cleavage and degradation. The absence of the mRNA template prevents protein synthesis, effectively silencing the gene.
• miRNA, on the other hand, operates via a slightly distinct mechanism. MiRNAs are encoded in the genome and produced endogenously within the cell. They are transcribed into primary miRNA transcripts, which undergo DICER processing to become mature miRNA molecules. Mature miRNAs associate with the RISC and direct it to complementary-sequenced target mRNA molecules. However, unlike siRNA, which cleaves mRNA, miRNA chiefly inhibits translation. It accomplishes this by preventing the ribosome from initiating protein synthesis by inhibiting the binding of translational factors to the mRNA.
• Both siRNA and miRNA mechanisms are referred to collectively as RNA interference because they both aim to silence or downregulate gene expression. This process enables cells to defend themselves from pathogens and modulate their own gene expression profiles. The formation of the RISC and DICER complexes is a crucial step in the RNAi pathway, as it enables the correct loading and guidance of small RNA molecules to their mRNA targets.
• Overall, RNA interference-mediated gene silencing is a potent instrument for molecular biology research and has significant therapeutic potential in a variety of fields, including gene therapy and the development of novel treatments for genetic diseases and viral infections.

Transcriptional gene silencing

• Transcriptional gene silencing is a well-established pathway for regulating gene expression through epigenetic factors. Epigenetic modifications, including DNA methylation, histone modifications, and chromatin remodeling, play a crucial role in rendering genes inactive.
• One of the most well-known mechanisms for gene silencing is DNA methylation. In this process, an enzyme called SAM (S-adenosyl methionine) adds methyl groups to the CpG regions of DNA, leading to gene inactivation. Methylation of DNA prevents the binding of transcription factors and other proteins involved in gene expression, effectively shutting down gene transcription and protein synthesis.
• Histone modifications also contribute to transcriptional gene silencing. Histones are proteins that interact with DNA to form a complex called a nucleosome. Histone modification involves the addition or removal of chemical groups, such as acetyl or methyl groups, on specific amino acids within histones like H2A, H2B, H3, and H4. These modifications can alter the structure of chromatin and influence gene expression. For example, the addition of certain chemical groups can cause the DNA to become tightly packed and condensed, resulting in the formation of heterochromatin. This condensed chromatin structure makes it difficult for transcriptional machinery to access the DNA, leading to transcriptional inactivation and gene silencing.
• Chromatin remodeling is another process involved in transcriptional gene silencing. It refers to the rearrangement of nucleosomes and changes in the chromatin structure to regulate gene accessibility. Chromatin remodeling complexes can alter the position or composition of nucleosomes, making it more challenging for transcription factors and other proteins to access the DNA sequence. By compacting the DNA and preventing the binding of transcriptional factors, chromatin remodeling contributes to gene silencing and the inhibition of protein synthesis.
• Overall, transcriptional gene silencing mediated by epigenetic factors such as DNA methylation, histone modifications, and chromatin remodeling provides a mechanism for long-term gene regulation. These epigenetic modifications lead to the tight packaging of DNA, making it inaccessible to transcriptional machinery and resulting in the silencing of gene expression. Understanding these mechanisms is crucial for unraveling the complexities of gene regulation and their implications in various biological processes and diseases.

Transposons in gene silencing

• Transposons, also known as transposable elements, are mobile genetic elements that have the ability to move within a genome. They can transpose or “jump” from one location to another, causing changes in the genetic sequence of an organism.
• There are two main types of transposon systems: DNA transposons and retrotransposons. DNA transposons are commonly found in prokaryotes, while retrotransposons are prevalent in eukaryotes. These transposon systems utilize different mechanisms for transposition, but both can contribute to gene silencing.
• Transposable elements have been found to be involved in gene silencing processes. When a transposon jumps into an active gene, it inserts itself into the gene’s DNA sequence. This insertion disrupts the normal gene structure and introduces additional genetic material that is not part of the original gene. As a result, the gene is unable to function properly, and its ability to undergo translation, the process by which proteins are synthesized, is impaired.
• In recent years, the concept of using transposons in genetic engineering has emerged. One example is the “sleeping beauty” transposon system, which has been harnessed as a tool to manipulate gene expression. The sleeping beauty transposon system involves the use of a synthetic transposon derived from a dormant transposon found in fish. This system allows researchers to introduce or remove specific genetic sequences from an organism’s genome, thereby modulating gene expression.
• The utilization of transposons in genetic engineering offers a versatile approach to study gene function, investigate gene regulation mechanisms, and potentially develop therapeutic applications. Transposons can be used to introduce new genetic material into specific genomic regions or disrupt target genes, allowing scientists to explore the effects of gene silencing and understand the functional consequences of gene expression modulation.

Antisense oligonucleotides

• Antisense oligonucleotides are a method used to silence specific genes by designing short nucleotide sequences that are complementary to the target mRNA. This technique, known as antisense oligonucleotide therapy, allows for the selective inhibition of gene expression.
• The concept of using antisense oligonucleotides to modulate gene expression was first reported by Paul Zamecnik and Mary Stephenson in 1978. They demonstrated that by introducing synthetic RNA molecules that are complementary to the target mRNA, it was possible to interfere with the translation process and inhibit protein synthesis.
• Antisense oligonucleotides work by binding to the complementary region of the target mRNA. Depending on the specific design and mechanism, they can achieve gene silencing through different approaches. One common strategy is to utilize the enzyme RNase H, which recognizes RNA-DNA hybrids and degrades the RNA component. When the antisense oligonucleotide hybridizes to the target mRNA, the RNA-DNA duplex is recognized by RNase H, leading to cleavage and degradation of the mRNA molecule. As a result, the mRNA cannot serve as a template for protein synthesis.
• Alternatively, antisense oligonucleotides can block translation by other means. For example, they can interfere with the assembly of the translational machinery or prevent the ribosome from initiating protein synthesis on the target mRNA. By blocking translation, the antisense oligonucleotides prevent the synthesis of the corresponding protein, effectively silencing the gene.
• The traditional term used to describe this approach is antisense RNA technology, highlighting the use of complementary RNA molecules to target specific mRNA sequences. Over the years, the field of antisense oligonucleotide therapy has advanced significantly, with improvements in oligonucleotide design, delivery methods, and chemical modifications to enhance stability and efficacy.
• Antisense oligonucleotides offer a promising avenue for targeted gene silencing and have been explored as potential therapeutics for various genetic disorders, including neurodegenerative diseases, cancer, and rare genetic conditions. Continued research and development in this field hold significant potential for the development of novel therapies that can selectively modulate gene expression and treat a wide range of diseases.

CRISPR-CAS9 gene silencing

• CRISPR-Cas9, known primarily for its gene editing capabilities, can also be used for gene silencing purposes.
• In the conventional activity of Cas9, a single-stranded guide RNA (gRNA) binds to the nuclease Cas9 and directs it to cleave the target nucleic acid sequence, effectively disrupting gene function. However, researchers have developed modified versions of Cas9 that can bind to the target sequence without cutting it. This modified Cas9, known as dCas9 (catalytically dead Cas9), lacks nuclease activity but retains its ability to bind to DNA or RNA.
• By using dCas9 in combination with a guide RNA, scientists can effectively silence or reduce gene expression. When dCas9 binds to the target gene, it hinders the recognition of the gene by RNA polymerase and other transcription factors. Consequently, the gene cannot be transcribed, and protein synthesis is inhibited.
• In addition to the use of dCas9, there are other naturally occurring gene silencing mechanisms in prokaryotes and eukaryotes that function as part of their defense systems to protect cells. These mechanisms include RNA silencing or suppression, which plays important roles in cellular metabolism and the regulation of cell cycles and division in mammals.
• In vitro studies utilize gene silencing techniques, including CRISPR-Cas9, to explore the functions and roles of specific genes. By silencing or reducing the expression of a particular gene, researchers can investigate the consequences and effects on cellular processes, development, disease mechanisms, and more. Gene silencing using CRISPR-Cas9 has various applications, including functional genomics research, target validation for drug discovery, and the development of potential therapies for genetic disorders and diseases.
• The versatility of CRISPR-Cas9, both in gene editing and gene silencing, has revolutionized the field of molecular biology and opened up new possibilities for understanding gene function and manipulating gene expression. Ongoing advancements in CRISPR technology continue to expand its applications and potential for scientific and therapeutic breakthroughs.

Applications of gene silencing

Gene silencing has a wide range of applications in various fields, including genetic engineering, medical research, and agriculture.

• In genetic engineering, gene silencing techniques are employed to construct transgenic organisms or genetically modified plants. This allows scientists to modify the genetic makeup of economically important plants, enhancing their traits such as yield, nutritional content, and resistance to pests or diseases.
• In the medical field, gene silencing is utilized to study genes associated with cancer, infectious disorders, and other genetic diseases. Overexpression of certain genes can contribute to the development of cancer, and gene silencing techniques using small hairpin RNA (shRNA) or microRNA (miRNA) have been employed to selectively silence these genes. This approach aims to inhibit the expression of cancer-promoting genes and potentially halt tumor growth.
• Gene silencing has also shown promise in the treatment of infectious diseases such as HIV. By targeting the viral RNA gene using small interfering RNA (siRNA), researchers can disrupt viral replication and reduce viral load. Trials are underway to assess the efficacy of this technique for treating HIV and hepatitis infections.
• In addition to cancer and infectious diseases, gene silencing methods are being explored as potential treatments for a range of other conditions. These include asthma, cystic fibrosis, chronic obstructive pulmonary disease (COPD), hepatitis B, hepatitis C, chronic myeloid leukemia, and neurodegenerative disorders. The ability to selectively silence disease-associated genes opens up possibilities for developing targeted therapies and gaining insights into disease mechanisms.
• In agriculture, gene silencing through RNA interference (RNAi) techniques is utilized to create genetically engineered plant species. This enables the development of crops with reduced toxin production or enhanced resistance to pests, thereby improving crop yields and reducing the need for chemical pesticides.

The applications of gene silencing extend beyond these specific fields. It is used in agribiotechnology, microbiology, food processing technology, and other scientific disciplines for various purposes. Gene silencing provides a powerful tool for understanding gene function, investigating disease mechanisms, developing novel therapeutics, and improving agricultural practices, leading to advancements in multiple scientific and industrial sectors.

The process of artificial gene silencing

Artificial gene silencing is a process used in genetic engineering to deactivate or reduce the expression of specific genes in an organism. By interfering with gene function, scientists can modify traits or reduce undesirable characteristics. The process typically involves several common steps, and we can explore the process of artificial gene silencing using the example of asparagus and the inactivation of the ASP5 gene.

Asparagus is known to contain a toxin called spirostanol saponins. To disrupt the function of a gene involved in the metabolism of these toxins, specifically the ASP5 gene, artificial gene silencing techniques can be employed.

One method of gene silencing is RNA interference (RNAi), which utilizes small RNA molecules to inhibit gene expression. In this case, short double-stranded RNA molecules, known as small hairpin RNA (shRNA), are constructed artificially. These shRNAs are typically 17 to 20 nucleotides long and designed to target and bind specifically to the ASP5 gene.

The constructed shRNA is then inserted directly into the plant cells using a technique called electroporation. Electroporation involves applying brief electrical pulses to create temporary pores in the cell membrane, allowing the entry of the shRNA molecules.

Alternatively, a gene for microRNA (miRNA) specific to the mRNA of the ASP5 gene can be inserted into a plasmid. The plasmid, carrying the miRNA gene, is transferred to Agrobacterium, a bacterium commonly used as a tool in genetic engineering. The Agrobacterium is then used to infect the target plant. Once inside the plant cell, the gene for microRNA inserts itself into the plant genome and produces miRNA molecules that can cleave the mRNA of the ASP5 gene.

The resulting miRNA molecules reduce the expression of the ASP5 gene, leading to a decrease in the production of toxins. Each time the miRNA binds to the ASP5 gene, it prevents it from transcribing and producing proteins involved in toxin metabolism.

In another method, the overall process remains similar, but instead of inhibiting gene expression, the ASP5 gene is cleaved or mutated to make it inactive. However, completely inactivating the gene may not be beneficial for the plant, as genes often have multiple functions. In the case of toxins produced by a snake, for example, they might be harmful to humans but serve as essential digestive enzymes for the snake.

To address this, the approach is to reduce the expression of the gene rather than making it entirely inactive. By regulating the expression level, scientists can control the production of toxins while still maintaining the gene’s other beneficial functions.

Through various approaches, scientists can create transgenic plants, animals, and model organisms to silence different genes. Artificial gene silencing techniques have a wide range of applications in genetic engineering and can be used to modify traits, enhance desirable characteristics, or reduce the expression of harmful genes in various organisms.

Gene knockdown vs gene silencing

Gene knockdown and gene silencing are two distinct techniques used in genetic research, often mistaken for being similar. While both techniques involve manipulating gene expression, there are important differences between them.

Gene knockdown refers to the process of reducing or inhibiting the expression of a specific gene. This technique aims to disrupt the normal function of the gene, preventing the synthesis of the corresponding protein. By interfering with the gene’s activity, gene knockdown can be used to halt the production of faulty or unwanted proteins. However, it does not completely eliminate gene expression. The gene may still produce some level of protein, albeit at a reduced rate.

On the other hand, gene silencing is a broader term that encompasses various methods of reducing gene expression. In gene silencing, the goal is to render the gene partially inactive rather than completely inactive. This means that the gene’s expression is diminished, leading to a reduction in the amount of protein formation. Gene silencing techniques can include RNA interference (RNAi), where small RNA molecules bind to and degrade the mRNA of the target gene, or epigenetic modifications that alter the gene’s accessibility to transcription machinery. The result is a decrease in overall gene expression and protein synthesis, but the gene is not entirely silenced or eliminated.

It is essential to avoid confusion between gene knockdown and gene silencing, as they have different implications for gene expression. Gene knockdown involves disrupting the normal function of the gene and preventing protein formation, while gene silencing aims to reduce gene expression without completely inactivating the gene. These techniques are utilized in different contexts and serve distinct purposes in genetic research.

FAQ

References

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• Bhardwaj, R., Ohri, P., Kaur, R., Rattan, A., Kapoor, D., Bali, S., … Singh, R. (2014). Gene Silencing. Emerging Technologies and Management of Crop Stress Tolerance, 209–228. doi:10.1016/b978-0-12-800876-8.00010-2
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