What Is an shRNA Screen and How Does It Work?

Functional genomics is a field of biology that aims to understand what specific genes do. A genetic screen is an approach designed to investigate thousands of genes at once to determine their roles in a biological process. An shRNA screen is a specialized type of genetic screen that uses small hairpin RNA (shRNA) to systematically reduce the activity of individual genes. This process, called gene silencing, allows researchers to observe how turning down a specific gene affects a cell, providing clues about that gene’s function.

The Mechanism of Gene Silencing

An shRNA screen utilizes a natural cellular process called RNA interference (RNAi), which cells use to regulate gene activity. An shRNA molecule is a synthetically created piece of RNA with a tight hairpin loop structure. When this shRNA is introduced into a cell, usually delivered by a disabled virus, it is processed by the cell’s machinery. Inside the cytoplasm, an enzyme called Dicer recognizes the hairpin shape and cuts the loop, creating a shorter, double-stranded RNA molecule. This smaller molecule is then loaded into a multi-protein machine called the RNA-induced silencing complex (RISC).

The RISC complex separates the two RNA strands and uses one as a guide. This guide strand directs the RISC to find and bind to messenger RNA (mRNA) molecules with a matching sequence. The mRNA is the cell’s working copy of a gene’s instructions for building a specific protein. Once the RISC complex binds to the target mRNA, it destroys it, preventing the cell from making the protein. This “silences” the gene without altering the cell’s underlying DNA.

Executing a High-Throughput Screen

The process begins with an shRNA library, a collection of molecules each designed to silence one specific gene. A comprehensive library can target nearly every gene in an organism’s genome. These libraries are packaged into viral vectors, like lentiviruses, for efficient delivery into a large population of cells. The goal is for each cell to take up just one shRNA from the library, creating a diverse population where each cell has a different gene silenced.

After treatment, the cells are subjected to a “selective pressure,” such as exposure to a new drug or nutrient deprivation. In a cancer drug screen, for example, most cells might die when exposed to the compound. However, cells where a specific gene has been silenced might survive, while silencing a different gene might make a cell more sensitive. Identifying which cells survive or die helps pinpoint genes involved in the cell’s response.

Pooled Versus Arrayed Screening Formats

Scientists perform shRNA screens in two main formats: pooled or arrayed. The choice depends on the experiment’s goals, scale, and available resources.

In a pooled screen, cells with different shRNAs are grown together in a single mixture. After applying selective pressure, the surviving cells are collected. Scientists then use high-throughput sequencing to identify and count the shRNA sequences in the surviving population’s DNA. This method is efficient for screening an entire genome to discover genes involved in a process like drug resistance.

The arrayed screen format is more organized. Cells are physically separated into individual wells of multi-well plates, and a single, known shRNA is delivered to the cells in each well, meaning the gene being silenced in any given well is already identified. The effect of the gene silencing is then measured on a well-by-well basis, often using automated microscopy to assess cell health. This format is more labor-intensive and expensive but provides direct data linking a specific gene to an outcome.

Interpreting Results and Identifying Hits

In a pooled screen, analysis focuses on changes in the representation of each shRNA. Scientists look for “enriched” shRNAs, which are found more frequently in surviving cells compared to the starting population. Enrichment suggests that silencing the gene provided a survival advantage. They also look for “depleted” shRNAs, which are less frequent, implying the target gene is needed for survival.

For an arrayed screen, interpretation is more direct. The outcome in each well, such as cell viability, is measured and compared to control wells where no gene was silenced. A “hit” is identified when a well shows a result that is statistically different from the control. For example, if cells in a specific well show significantly higher survival after drug treatment, the gene targeted in that well is considered a hit.

The result from either screen is a list of candidate genes, or “hits,” that appear to influence the biological process. These hits are not final conclusions but strong leads that require further investigation to validate their function and understand the mechanism by which they work.

Comparison to CRISPR-Based Screens

shRNA technology is often compared to CRISPR-based screens, a more recent method for studying gene function. The primary difference is their mechanism. An shRNA screen induces gene “knockdown,” which reduces the amount of protein produced from a gene but does not eliminate it. CRISPR is used for gene “knockout,” making a permanent change to the cell’s DNA to completely disable the gene.

This distinction has implications. Because shRNA’s effect is a reduction rather than a complete loss, it can be useful for studying genes required for a cell’s basic survival, as a complete knockout would kill the cell. The incomplete nature of knockdown can reveal functions that would be missed with a knockout approach.

shRNA screens can have “off-target” effects, where an shRNA molecule inadvertently silences unintended genes due to sequence similarity. CRISPR systems are more specific, with fewer off-target effects, leading to cleaner data. The decision to use an shRNA or a CRISPR screen depends on the scientific question, as each tool has distinct advantages and they are often seen as complementary approaches.