A Fluorescence-Activated Cell Sorting (FACS)-based CRISPR screen is a high-throughput technique used to systematically identify genes that influence a specific cellular process. This method combines the gene-editing of CRISPR-Cas9 with the single-cell analysis of FACS. By knocking out thousands of genes across a population of cells and then sorting those cells based on a fluorescent signal, researchers can discover which genes are responsible for controlling the measured output.
Key Components of the System
This screening method relies on three interacting components. The first is the CRISPR-Cas9 system, which consists of the Cas9 nuclease, an enzyme that cuts DNA like molecular scissors, and a guide RNA (gRNA) that directs the enzyme to a precise genomic location. When the gRNA binds to its matching DNA, Cas9 creates a double-strand break. The cell’s natural repair process often fixes this break imperfectly, resulting in the functional knockout of the targeted gene.
The second component is a pooled gRNA library, a mixture of thousands of unique gRNA sequences constructed in lentiviral vectors. These are viruses engineered to deliver genetic material into cells. Each viral particle contains a single gRNA designed to target one gene, allowing for a parallel experiment where each cell in a population has a different gene knocked out.
The final component is a reporter cell line modified to have a fluorescent output linked to a biological process. A common strategy is to insert a gene for a fluorescent protein, like Green Fluorescent Protein (GFP), into the cells. The expression of GFP is controlled by a genetic element that responds to a specific cellular event, such as a signaling pathway being activated.
The Step-by-Step Experimental Process
The workflow begins by introducing the pooled gRNA library into the reporter cells, often via lentiviral transduction. The process is calibrated to a low multiplicity of infection (MOI) to ensure each cell receives only one unique gRNA. This one-to-one correspondence is necessary for linking a gene knockout to a phenotype. Afterward, an antibiotic may be used to eliminate cells that did not incorporate the viral vector.
After the gRNA library is integrated, the cells are cultured for seven to fourteen days. This incubation allows time for the Cas9 enzyme to edit the DNA at the site specified by the gRNA. It also provides a window for the pre-existing protein encoded by the target gene to degrade. This ensures the genetic change manifests as a functional change, altering the fluorescent reporter’s signal.
The next step is sorting the cells using FACS. The population of cells is directed into a fluid stream, passing single-file through lasers that excite their fluorescent proteins. Detectors measure the light emitted from each cell, and based on this intensity, the instrument applies an electrical charge to the droplet containing the cell.
This charge physically separates the cells into different collection tubes. The operator defines gates, or specific windows of fluorescence intensity, to isolate subpopulations of interest. For example, cells exhibiting the top 5-10% of fluorescence are collected into a “high bin,” while cells with the bottom 5-10% of fluorescence are collected into a “low bin.” The large population of cells with intermediate fluorescence is discarded.
Analyzing the Results
Analysis begins by extracting genomic DNA from the sorted high and low fluorescence populations. This DNA contains the unique gRNA sequences that were integrated into each cell’s genome. Polymerase Chain Reaction (PCR) is then used to amplify the gRNA-encoding regions, creating millions of copies from each bin for identification.
The amplified gRNA sequences are then analyzed using Next-Generation Sequencing (NGS). This technology determines the DNA sequence of millions of fragments in parallel. The primary output is a data table that quantifies the abundance of each gRNA in both the high and low bins, essentially counting how many times each gRNA appeared in each population.
The analysis involves a computational comparison of gRNA frequencies between the two bins. Statistical methods are used to calculate an enrichment or depletion score for every gRNA. A gRNA found more frequently in the low-fluorescence bin suggests that knocking out its target gene caused a decrease in the signal, identifying the gene as a positive regulator. Conversely, a gRNA enriched in the high-fluorescence bin indicates its gene knockout increased the signal, identifying it as a negative regulator. This process transforms raw sequencing counts into a ranked list of genes that influence the phenotype.
Applications and Research Discoveries
FACS-based CRISPR screens can answer a wide range of biological questions by mapping complex cellular processes. One application is in deciphering cellular signaling pathways. For instance, if a fluorescent reporter is designed to light up when a specific pathway is active, a screen can identify genes that positively or negatively regulate that pathway, revealing previously unknown components.
This technique is also useful for identifying mechanisms of drug resistance in cancer research. Researchers can treat cancer cells with a therapeutic agent and use a fluorescent marker linked to cell survival. The screen can then pinpoint which gene knockouts enable cells to survive the drug treatment, uncovering genetic vulnerabilities and potential new targets for combination therapies.
Another application involves studying the regulation of proteins on the cell surface. Instead of an internal reporter, cells are stained with a fluorescently labeled antibody that binds to a specific surface protein. Using FACS, cells are sorted based on this protein’s abundance on their surface. This approach identifies genes involved in the protein’s production, transport, and stability, providing a comprehensive view of its life cycle.