Cell-to-cell communication is vital for the proper functioning of tissues in our bodies. Every action, from immune responses to brain activity, involves cells “talking” to each other, exchanging signals that guide their behavior. Understanding these interactions can help us figure out how diseases like cancer or neuroinflammation occur and how they can be treated. But studying these complex communications at a large scale has been a challenge—until now.
Researchers at Harvard Medical School have developed a new technique called SPEAC-seq (Systematic Perturbation of Encapsulated Associated Cells followed by sequencing), which allows scientists to investigate how cells interact with each other on a genome-wide scale. This technique combines advanced tools like CRISPR-Cas9, which can edit genes, and microfluidics, a technology that controls tiny amounts of fluids to work with cells more precisely. By using SPEAC-seq, scientists can observe how thousands of genes influence cell-to-cell communication in a much more efficient way than traditional methods.
How SPEAC-seq Works
The main idea behind SPEAC-seq is to encapsulate cells in tiny droplets and use genetic tools to turn off (or inactivate) specific genes in those cells. Then, by using fluorescent reporter circuits—essentially glowing signals that indicate specific cellular responses—scientists can see how these gene edits impact the way cells interact. For example, they might look at how one cell sends an inflammatory signal to another and how gene changes in the first cell affect this communication.
One of the big advantages of SPEAC-seq is that it doesn’t require scientists to already know which genes or interactions they want to study. This opens the door to discovering new, unknown pathways and regulators of cell communication.
Overview of the SPEAC-seq workflow
Microfluidic PDMS devices are prepared following photolithography techniques using SU-8 negative photoresist (Steps 1–5). PDMS devices are bonded to glass slides using oxygen plasma and the resulting channels are rendered hydrophobic with Aquapel treatment (Steps 6–14). The genome-wide CRISPR knockout screening library is amplified (Steps 15–19), packaged into lentiviral particles (Steps 20–26) and cell populations to be perturbed are stably transduced (Steps 27–28). Reporter cells are prepared as needed (Step 29). In our application of SPEAC-seq, primary microglia were transduced with genome-wide CRISPR knockout libraries, and primary astrocytes from p65EGFP NF-κB reporter mice were prestimulated at subthreshold cytokine concentrations to prime the reporter circuit. Co-encapsulation of a CRISPR screening cell and a reporter cell in single droplets is performed with a microfluidic device (Steps 29–36). Droplet emulsion containing co-encapsulated cells is collected and cells interact with each other over 24 h (Steps 37–38). Cocultured droplets are reinjected into a second microfluidic device operated in combination with lasers, fluorescence detectors, a field-programmable gate array (FPGA) and a high-voltage amplifier for fluorescence detection and real-time droplet sorting (Steps 39–45). Reporter fluorescence resulting from NF-κB activation (act.) is detected within microfluidic system and droplets are positively sorted via dielectrophoresis (Step 45). Positive and negative droplets are collected, and emulsions are chemically broken with perfluorooctanol (PFO) for cell recovery (Steps 46–48). gRNA sequences are amplified by PCR and libraries are prepared for sequencing (Steps 49–57).
A Real-World Example: Studying Brain Inflammation
The researchers tested this technique by focusing on neuroinflammation, which involves the activation of brain cells called microglia and astrocytes. In brain inflammation, these cells communicate in ways that can either help protect the brain or contribute to damage, depending on the signals they exchange. Using SPEAC-seq, the team screened a huge library of CRISPR-Cas9 inactivations to find out which genes in microglia influence the activation of astrocytes—specifically looking at the NF-κB signaling pathway, a key player in inflammation.
Their findings revealed thousands of genes in microglia that regulate how they activate astrocytes, offering new insights into how the brain responds to injury or disease. These discoveries could eventually lead to new treatments for conditions like Alzheimer’s or multiple sclerosis, where inflammation plays a big role.
Why SPEAC-seq is Important
SPEAC-seq is versatile—it can be adapted to study interactions between different types of cells, in various contexts, and using many different kinds of functional signals. This flexibility means it could be applied to cancer research, immune system studies, or any biological process where cell communication is critical. And it’s efficient, too—researchers can run a genome-wide screening in less than two weeks.
Overall, SPEAC-seq represents a powerful new tool for exploring the complex web of cell interactions in ways that were previously impossible. By giving scientists the ability to screen for genetic regulators of cell communication on a large scale, it opens up exciting possibilities for understanding—and eventually treating—a wide range of diseases.