For years Yale researchers David Breslow and Mustafa Khokha have worked together with a similar challenge in their sights – trying to capture the interplay between certain genes and the pediatric developmental disorders they cause.
In their latest collaboration, they hit pay dirt.
Using a new CRISPR screening technology developed by the Breslow lab, the researchers gained an unprecedented view into cellular sub-structures known as cilia and how ciliary defects are linked to different diseases. Along the way, they also discovered a microprotein that is crucial in normal embryonic development.
The new screening method — which is described in the journal Developmental Cell — could speed up discovery of genes linked to human developmental disorders and possibly other disease processes.
“Our method makes today’s newer and more powerful genetic screening technologies compatible with microscopy in a way that lets you assess cellular activities in finer detail, and it’s much easier to use,” said Breslow, an associate professor of molecular, cellular, and developmental biology in Yale’s Faculty of Arts and Sciences and a member of the Yale Cancer Center. “You don’t need to be an expert in microscope optics or software programming.”
When their latest collaboration began, Khokha was a professor of pediatrics and genetics at Yale School of Medicine. He has since become a professor of pediatrics at Cedars-Sinai Medical Center.
Breslow and Khokha originally met years ago when they were both part of the Yale Cilia Group, a consortium of research labs with shared interest in cilia, the tiny, hair-like structures found on many cells that act as a sort of antennae, allowing cells to receive signals from their environment and other cells.
This movie was generated using a technique called optical coherence tomography to visualize the movement of particles during frog embryonic development. This movement is driven by specialized cells with motile cilia. In the first part of the movie, a normal embryo is shown with rapid particle movement. The second part of the video shows an embryo in which the newly discovered ciliary protein tzmp1 (transition zone microprotein 1) was genetically inactivated, leading to loss of functional motile cilia and the absence of particle movement.
For the new work, their team focused on cilia. Mutations in cilia genes cause a host of pediatric disorders that impact the formation of bodily systems like the skeleton, heart, and brain, past research has shown.
To better understand the factors driving these outcomes, the researchers developed a new screening platform that allows them to search the entire genome for genes that affect cilia in specific ways. Importantly, the approach can also be adapted to study other organelles or diseases, not just cilia. They created the new methodology by merging CRISPR gene editing technology, automated microscopy, an AI image analysis tool, and a trick using UV light to mark individual cells of interest.
This approach allowed Breslow and his team to scan millions of cells, looking for mutant cells with visibly altered cilia.
“In the past, we’ve been able to really only look at cilia from one angle,” Breslow explained. “This new technology allows us to see cilia from lots of different angles, and we get a more complete picture of their biology and their functions.”
Using the new system, the team discovered hundreds of genes that control how cilia form and function. They also found a microprotein that sits at a key site within cilia and is essential for both cilia formation and for normal development in frog embryos, which suggests a potential link to human genetic disease as well.
“Going forward, we’ll be using this technology in my lab,” Breslow said. “It’s going to increase our understanding of the ways cilia contribute to disease, and it can be deployed by other researchers investigating all kinds of cellular processes and disorders.”
Other researchers on the project included associate research scientist Jingbo Sun and postgraduate researcher Irem Sude Atis, both in Yale’s Department of Molecular, Cellular and Developmental Biology; and Stéfany L. L. Empke a former Yale postdoctoral researcher who is now a postdoctoral scientist at Cedars-Sinai Medical Center, where she continues to work with Khokha.
Grants from the National Institutes of Health supported this research.
