A new genetic screening method allows researchers to efficiently modulate individual genes across entire tissues and provides new insights into human development.
Human neural tube organoid morphogenesis after ZIC2 knockdown. Image credit: Huang, Anand et al. (CC BY 4.0)
The research, published previously as a Reviewed Preprint in eLife and appearing today as the final Version of Record, is described as a landmark study by eLife’s editors. They go on to say in their assessment of the work that “this technical tour de force is exceptional and one of the first studies to reveal new knowledge on human development through embryo models”.
Performing genetic screening in humans and other animals poses ethical and practical challenges. An alternative is to use organoids – three-dimensional mini-organs – developed from human pluripotent stem cells (hPSCs). These are cells that have the potential to develop into any type of tissue. However, attempts to knock out or deplete individual genes in these models usually lead to a mosaic-like patchwork effect across the organoid, making it impossible to study tissue-wide structural development (or morphogenesis).
“The ability to perform single-gene perturbations in robust in vitro models of human development is essential for dissecting the mechanisms that drive human embryonic morphogenesis,” says co-first author Roya Huang, who at the time of the study was in senior author Sharad Ramanathan’s lab in the Department of Molecular and Cellular Biology, Harvard University, US, and is now a Postdoctoral Fellow in the Department of Molecular and Cell Biology, University of California, Berkeley, US. “Until now, this goal has been limited by variability in organoid morphogenesis and the lack of scalable methods for uniform single-gene perturbations. We have developed a method to generate and apply high concentrations of virus directly to stem cells in small volumes, resulting in a streamlined gene editing approach that can uniformly knock down genes across an entire organoid.”
Existing CRISPR gene editing methods for whole tissue gene screening are limited by the time and cost of isolating and cultivating individually edited clones, whereas alternative non-clonal approaches that use viruses to introduce genes into cells rely on laborious and time-consuming virus concentration steps.
“Usually, CRISPR gene editing requires changing a circular piece of DNA – a plasmid – to contain a specific piece of genetic material, a process that usually takes several sequential steps,” explains co-first author Giridhar Anand, who was also in the Ramanathan Lab at the time of the study and is now a Postdoctoral Research Fellow at Memorial Sloan Kettering Cancer Center, New York, US. “We instead conducted multiple steps in parallel and performed a stringent DNA purification step at the beginning to ensure that a high proportion of the resulting plasmids were engineered correctly. This allowed us to skip the next stage in plasmid preparation, namely the time-consuming process of selecting and cultivating specific clones.” The resulting plasmids were then packaged to be delivered to organoid cells by a virus.
The next challenge for the team was to generate high concentrations of virus. They optimised growth of the virus in cell lines, finding that reducing the volume of the media that the cells grow in increased the yield of the virus. They also found that adding the virus at the same time as seeding stem cells on the growth plate improved the uptake of the virus compared to the standard method of seeding cells first and then adding the virus. This optimised method resulted in the uptake of the virus by almost all the hPSCs.
To achieve knockdown of multiple genes in a single experiment, the team developed a way to deliver different plasmids, each carrying a different piece of DNA, to separate colonies of cells on a microscope slide. These cells could then be differentiated into different types of organoids, each featuring a unique gene alteration in every cell.
To test their approach, the team then chose 20 genes thought to be involved in a crucial brain development step in which a flat neural plate closes into a neural tube. If this step does not occur during development, it causes a fatal birth defect called anencephaly where a baby is born without a properly folded front part of the brain (forebrain) and the thinking and coordinating part of the brain (cerebrum).
The team used their streamlined CRISPR method to produce plasmids for these 20 genes, plus a further 57 genes which are less strongly linked to neural development. They introduced these plasmids to hPSCs and then differentiated the cells into neural tube organoids.
After incubation, they stained the organoids for neural tissue markers and studied their shape under a microscope. They found that the knockdown of three genes called ZIC2, SOX11 and ZNF521 showed major neural tube closure defects. In the organoids with ZIC2 and SOX11 knockdown, the neural plates were fully open, whereas in the ZNF521 knockdown organoids there were multiple points of closure.
The team next studied single-gene expression data to see if there were other genes affected by the knockdown of these three main players. They identified a further subset of genes that were less active in the ZIC2 and SOX11 knockdown organoids, and more active in the ZNF521 organoids. When they tried to deplete these genes individually, no single gene resulted in the same neural tube closure 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, greatly reduces time and costs compared to clonal knockdown approaches, and can be performed in an academic laboratory setting, enabling tissue-wide single-gene perturbations at a scale that has not been previously feasible either in lab-grown organoids or in mammalian models,” says senior author Sharad Ramanathan, Principal Investigator and the Llura and Gordon Gund Professor of Neurosciences and of Molecular and Cellular Biology at Harvard University. “Our approach bridges a critical gap between the genetic study of traditional model organisms and human developmental biology, offering a path for new mechanistic insights and the discovery of therapeutic targets for neural tube defects and other congenital malformations.”
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