New genetic screening methods allow researchers to efficiently regulate individual genes across tissues, providing new insights into human development.
This study was previously published as a reviewed preprint. e-life And today, the paper, which appears as the final version of the record, has been described by eLife editors as groundbreaking research. In their review of the study, they continued: “This technical tour de force is exceptional, and one of the first studies to reveal new knowledge about human development through an embryonic model.”
Performing genetic screens in humans and other animals presents ethical and practical challenges. Another method is to use organoids (three-dimensional mini-organs) developed from human pluripotent stem cells (hPSCs). These are cells that can develop into all types of tissues. However, attempts to knock out or deplete individual genes in these models typically result in a mosaic patchwork effect throughout the organoid, making it impossible to study structural development (or morphogenesis) throughout the tissue.
“The ability to perform single gene perturbations in robust in vitro models of human development is essential for elucidating the mechanisms that drive human fetal morphogenesis,” said co-first author Roya Huang. At the time of the research, he was in the laboratory of senior author Sharad Ramanathan in the Department of Molecular and Cellular Biology at Harvard University, and is currently a postdoctoral researcher in the Department of Molecular and Cellular Biology. Biology, University of California, Berkeley, USA. “Until now, this goal has been limited by variability in organoid morphogenesis and the lack of scalable methods for uniform perturbation of single genes. We have developed a method to generate high concentrations of virus and apply it directly to stem cells in small quantities. The result is an efficient gene editing approach that can knock down genes uniformly throughout the organoid.”
Existing CRISPR gene editing methods for whole-tissue genetic screening are limited by the time and cost of isolating and culturing individually edited clones, while alternative non-clonal approaches that use viruses to introduce genes into cells rely on laborious and time-consuming virus enrichment steps.
“CRISPR gene editing typically involves modifying a circular DNA (plasmid) to contain specific genetic material, and the process typically requires several sequential steps,” explained co-first author Giridhar Anand. He was also a member of Ramanathan’s laboratory at the time of the research, and is currently a postdoctoral researcher at Memorial Sloan Kettering Cancer Center in New York, USA. “Instead, we performed multiple steps in parallel, first performing a rigorous DNA purification step to ensure that a high proportion of the resulting plasmids were manipulated correctly. This allowed us to skip the next step in plasmid preparation, the time-consuming process of selecting and culturing specific clones.” The resulting plasmids were packaged for delivery to organoid cells by virus.
The team’s next challenge was to generate high concentrations of the virus. They optimized virus growth in cell lines and found that reducing the amount of medium in which the cells were grown increased virus yield. They also found that adding virus at the same time as seeding stem cells on growth plates improved virus uptake compared to the standard method of seeding cells first and then adding virus. This optimized method resulted in virus uptake in nearly all hPSCs.
To achieve knockdown of multiple genes in a single experiment, the team developed a method to deliver different plasmids, each carrying a different piece of DNA, to isolate colonies of cells on a microscope slide. These cells can differentiate into different types of organoids, each characterized by unique genetic changes.
To test their approach, the team next selected 20 genes thought to be involved in a key brain development step in which the flattened neural plate closes into the neural tube. If this step does not occur during development, it causes a fatal birth defect called anencephaly, in which babies are born with the front part of the brain (forebrain) and the thinking and coordinating part of the brain (cerebrum) not folded properly.
Using a streamlined CRISPR method, the research team created plasmids for these 20 genes, plus an additional 57 genes that are less strongly associated with neurodevelopment. They introduced these plasmids into hPSCs and allowed the cells to differentiate into neural tube organoids.
After incubation, the organoids were stained for neural tissue markers and their shape studied under a microscope. They found that knocking down three genes called ZIC2, SOX11, and ZNF521 showed severe neural tube defects. In organoids with ZIC2 and SOX11 knockdown, the neural plate was completely open, whereas in organoids with ZNF521 knockdown, there were multiple closure points.
The research team then studied single-gene expression data to see if there were other genes affected by the knockdown of these three key players. They identified an additional subset of genes with low activity in ZIC2 and SOX11 knockdown organoids and high activity in ZNF521 organoids. When they tried to deplete these genes individually, no single gene caused the same neural tube defect. This suggests that ZIC2, SOX11, and ZNF521 normally control a combination of downstream genes to direct neural tube closure.
The platform we report here is scalable, significantly reducing time and cost compared to clonal knockdown approaches, can be performed in academic laboratory settings, and allows perturbation of single genes throughout tissues at a scale previously unfeasible in laboratory-cultured organoids or mammalian models. Our approach bridges a critical gap between traditional model organism genetic studies and human developmental biology, providing avenues for the discovery of new mechanistic insights and therapeutic targets for neural tube defects and other congenital malformations. ”
Sharad Ramanathan, Harvard University Senior author and Rula Gund and Gordon Gund Professor of Neuroscience and Molecular Cell Biology
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Reference magazines:
fan, RE, Others. (2026) Sequenced single gene perturbations identify drivers of human anterior neural tube closure.. e-life. DOI: 10.7554/eLife.108224.3. https://elifesciences.org/articles/108224
