Understanding how a single genetic mutation can alter the shape of a developing organ has long posed a challenge for developmental biologists. While researchers can readily study the effects of gene mutations on individual cells, uncovering how those mutations influence the coordinated movements of thousands of cells that sculpt tissues and organs has been much more difficult.
Now, researchers from the lab of MCB Professor Sharad Ramanathan have developed a new approach that makes those questions far easier to investigate.
In a study published in eLife, former MCB graduate students Roya Huang and Giridhar Anand, who share co-first authorship and are now postdoctoral fellows at the University of California, Berkeley, and Memorial Sloan Kettering Cancer Center, respectively, combined complementary expertise to develop a rapid, inexpensive method for introducing the same CRISPR gene perturbation into nearly every cell of a developing human organoid. Huang led the developments for perturbing cells at high efficiency with gene-targeting viruses, while Anand developed a rapid and streamlined cloning strategy, together making large-scale genetic screening practical and efficient. The work was completed in the Ramanathan lab.
The technique allows scientists to examine how individual genes influence the overall architecture of developing tissues rather than just the behavior of isolated cells.
“The goal of the project was to find individual genes that are necessary for the formation of proper organismal shape in humans,” Huang said. “The establishment of proper morphology involves the coordinated movements of hundreds of thousands of cells, so studying the role of a single gene in this process requires that all of those cells have to carry the same genetic perturbation.”
Human organoids—three-dimensional tissues grown from stem cells that mimic aspects of organ development—have become powerful models for studying human biology. Researchers commonly use pooled CRISPR screens in organoids to investigate how genes affect individual cells. However, because each genetic perturbation reaches only a small fraction of cells, those approaches are poorly suited for studying large-scale tissue movements during development.
Huang explained that the team’s method overcomes this limitation by introducing a single CRISPR guide RNA with exceptionally high efficiency, allowing an average of about 93 percent of cells within an organoid to receive the same genetic perturbation.
“That allows us to understand how that single gene affects the morphology, or the entire shape, of the organoid, rather than the outcome of a particular cell,” Huang said. The approach also removes several practical barriers that have limited large-scale developmental genetics experiments.
Existing methods often require researchers to spend weeks or months generating individual genetically engineered stem cell lines or rely on specialized robotic systems that are costly and unavailable to many laboratories.
“This method is unique in that it’s very fast, it’s very cheap, and it’s very accessible to any lab with a standard tissue culture setup,” Huang said. “Because there is no selection step required to produce a comprehensively perturbed cell population, it also produces consistent starting conditions that lead to robust and reproducible organoids.”
While the study introduces a broadly applicable experimental platform, it also demonstrates its biological power by tackling one of the earliest and most important events in human development: neural tube closure.
During early embryonic development, the tissue that will become the brain and spinal cord begins as a flat sheet of cells before folding into a tube. Failures in this process can lead to severe congenital malformations, including spina bifida and anencephaly, a devastating defect in which the forebrain fails to close properly.
Because anencephaly is typically lethal before or shortly after birth, researchers have had limited opportunities to identify the genes responsible through traditional human genetic studies.
Using their new screening platform, Huang, Anand, and colleagues created an in vitro model of anterior neural tube closure and systematically tested 77 candidate genes for their roles in early human forebrain development. The researchers identified three genes—ZIC2, SOX11, and ZNF521—that proved essential for proper neural tube closure in their organoid model.
“We built an anterior neural tube model and screened these genes to determine their requirement for proper neural tube closure in the forebrain,” Huang said. “We found that three genes were essential for this process in our in vitro model.”
The findings provide new insight into the genetic mechanisms that govern early human brain development and establish a framework for investigating congenital birth defects that are otherwise difficult to study.
Huang and Anand hope the platform will find applications far beyond neural tube development.
“We think this system would be applicable to any other organoid morphology system or questions involving congenital malformations,” Huang said. “We hope people use it to discover other potential regulators of morphological processes during human development.”
Although Huang and Anand have since moved to new labs for postdoctoral training, the work reflects years of collaboration in the Ramanathan Lab, combining method development with developmental biology to create a tool that other laboratories can readily adopt.
By making large-scale genetic perturbation experiments faster, simpler, and more affordable, the researchers hope the new approach will accelerate efforts to understand how genes shape human development—and why that process sometimes goes awry.
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