Gene knockdown is a molecular biology technique that reduces the expression of a specific gene within a cell or organism. This reduction allows scientists to investigate gene function by observing changes in cellular processes or organismal traits. It also offers therapeutic potential by dampening the activity of genes implicated in diseases.
Unraveling How Gene Knockdown Works
Gene knockdown interferes with messenger RNA (mRNA) molecules, which carry genetic instructions from DNA to the cell’s protein-making machinery. A prominent mechanism is RNA interference (RNAi), a natural cellular process. In RNAi, small, double-stranded RNA molecules, such as small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs), guide a protein complex called the RNA-induced silencing complex (RISC) to a specific mRNA target.
Once the siRNA or shRNA is loaded into RISC, one strand (the guide strand) directs the complex to locate and bind to complementary mRNA sequences in the cytoplasm. Argonaute 2 (Ago2), a key RISC component, then cleaves and degrades the targeted mRNA. This degradation prevents the mRNA from being translated into a protein, reducing the amount of that protein.
Another mechanism involves antisense oligonucleotides (ASOs), synthetic, single-stranded DNA or RNA molecules, typically 15-25 nucleotides long. ASOs bind to complementary mRNA sequences, forming an RNA-DNA hybrid. This binding triggers RNase H to cleave the RNA strand within the hybrid, degrading the target mRNA. ASOs can also inhibit protein production by physically blocking ribosome binding or altering mRNA maturation.
Tools and Techniques for Gene Knockdown
Scientists employ several tools and techniques for gene knockdown. Small interfering RNAs (siRNAs) and antisense oligonucleotides (ASOs) can be directly introduced into cells. SiRNAs are often delivered via transfection. ASOs, as single-stranded synthetic nucleic acids, rely on chemical modifications for cellular uptake. However, direct introduction often provides only transient knockdown.
For stable, long-term gene knockdown, short hairpin RNAs (shRNAs) are often utilized. shRNAs are expressed from DNA constructs, often delivered into cells using viral vectors such as lentiviruses or adeno-associated viruses (AAVs). Once inside, these viral vectors integrate shRNA-encoding DNA into the host genome, allowing continuous production of shRNA molecules processed into siRNAs by cellular machinery.
CRISPR interference (CRISPRi) is a newer approach leveraging a modified CRISPR-Cas system. Instead of cutting DNA like traditional CRISPR-Cas9, CRISPRi uses a catalytically dead Cas9 (dCas9) protein. Guided by a guide RNA to a gene’s DNA sequence, dCas9 physically blocks transcription, preventing gene expression into mRNA.
Why Gene Knockdown Matters
Gene knockdown plays an important role in basic scientific research, allowing investigators to understand individual gene function. By reducing a gene’s activity, researchers observe changes in cellular behavior, development, or organismal physiology, helping deduce the gene’s normal role within biological pathways.
This technique is useful in drug discovery and development. Gene knockdown helps identify and validate potential drug targets by identifying genes whose reduced activity leads to a desired therapeutic effect. It can also confirm the mechanism of action of new therapeutic compounds.
Gene knockdown offers potential for new treatments for genetic diseases. For instance, patisiran and givosiran are siRNA-based drugs approved for hereditary amyloidogenic transthyretin (hATTR) amyloidosis and acute hepatic porphyria by decreasing disease-causing protein expression. This approach offers therapeutic avenues for conditions like certain cancers and genetic disorders such as Huntington’s disease.
Gene Knockdown Compared to Gene Knockout
Gene knockdown and gene knockout are distinct techniques for studying gene function, differing in their impact on expression. Gene knockdown reduces gene expression, often resulting in a partial or temporary decrease in protein levels, while the gene remains present. This reduction can be transient or stable, depending on continuous production of the knockdown agent.
In contrast, gene knockout involves complete elimination or inactivation of a specific gene from an organism’s genome. This is achieved by disrupting the gene’s DNA sequence, often through gene-editing technologies like CRISPR-Cas9, leading to a complete absence of the corresponding protein. The effect is irreversible and inheritable, passed on to subsequent generations of cells or organisms.
The choice between these techniques depends on the research question. Gene knockdown is often preferred when studying essential genes, as a complete knockout might be lethal, preventing observation of specific phenotypes. It also allows for studying dose-dependent effects, as gene expression reduction can be modulated. Gene knockout, however, provides a clear view of a gene’s full contribution by completely removing its function, useful for understanding the comprehensive impact of its absence.
Important Considerations in Gene Knockdown
Despite its utility, gene knockdown has several practical considerations and limitations. A challenge is off-target effects, where the knockdown agent affects unintended genes. For siRNAs, this can occur if partial complementarity exists between the siRNA’s “seed region” and the 3′-untranslated regions of other mRNAs, leading to unintended repression. Researchers often use multiple siRNAs or shRNAs targeting the same gene to mitigate this, as different knockdown agents are unlikely to have the same off-target effects.
Many gene knockdown approaches, particularly those using directly introduced siRNAs, are transient, requiring repeated application to maintain reduced gene expression. This is a practical limitation in long-term studies or therapeutic applications. While viral vectors enable stable, long-term knockdown by integrating shRNAs into the genome, these methods introduce complexities, including potential immune responses and challenges in controlling expression levels.
Delivering knockdown agents to specific tissues or cells within a living organism presents difficulties. For instance, delivering siRNA duplexes is more challenging than single-stranded antisense oligonucleotides; lipid-based formulations are often used to target specific organs like the liver. Gene knockdown efficiency can also vary, meaning the desired reduction may not always be achieved consistently across all cells or experiments.