A team of researchers at the University of California, San Francisco (UCSF), and the National Institutes of Health (NIH) have developed a new CRISPR-based approach to investigating gene function in neurons that could significantly improve scientists’ ability to study brain diseases. The new method uses a CRISPR interference (CRISPRi) developed by the UCSF researchers as a platform for genetic screens in neurons derived from human induced pluripotent stem cells (iPSCs).
The researchers say that to their knowledge, the studies, described in Neuron, mark the first time that CRISPR technology has been used for large-scale screening in any differentiated human iPSC-derived cell types. “Prior to this study, there were significant limitations that restricted what scientists could do with human neurons in the lab,” said Martin Kampmann, PhD, associate professor in UCSF’s Institute for Neurodegenerative Diseases, and an investigator at the Chan-Zuckerberg Biohub. Kampmann is co-senior author of the researchers’ study, which is titled, “CRISPR Interference-Based Platform for Multimodal Genetic Screens in Human iPSC-Derived Neurons.”
Genetic variants and mutations are known to be associated with an increased risk for many neurological diseases, but technological bottlenecks have scuppered scientists’ ability to study how these genes cause disease. And until relatively recently there was no reliable way of obtaining human brain cells that could be used for complex lab experiments, explained Kampmann, who is also a member of the UCSF Weill Institute of Neurosciences. “It was possible to get neurons donated by patients who had undergone procedures that involve removing brain tissue to treat epilepsy or brain cancer. But these samples can only survive for a few days. You can’t perform experiments to probe gene function on short-lived neurons.”
With a lack of human brain cells for study, scientists were resigned to work with animal models of brain disease, but model non-human systems don’t necessarily recapitulate human neurobiology. The situation changed in 2006 with the development of induced pluripotent stem cell technology, through which normal adult cells, such as skin cells, could be deprogrammed back into multipotent stem cells, and then reprogrammed to differentiate into any cell type in the body, including neurons. It was hoped that the subsequent development of CRISPR-Cas9 gene-editing technology would provide the tools needed to allow scientists to study the genetic basis of neurological disorders in iPSC-derived neurons.
CRISR-Cas9 harnesses the Cas9 enzyme to snip double-stranded DNA at defined sites, but it was found that the system doesn’t work well with stem cells. “Stem cells have a very active DNA damage response,” Kampmann said. “When Cas9 produces even just one or two DNA cuts, it can lead to toxicity that causes the cells to die.” As the authors further stated, “… most previous CRISPR-based screens were implemented in cancer cell lines or stem cells rather than healthy differentiated human cells, thereby limiting potential insights into cell-type-specific roles of human genes … While CRISPR screens in cancer cells and stem cells have revealed numerous biological insights, we reasoned that screens in differentiated, non-cancerous cell types could elucidate novel, cell-type-specific gene functions.”
Kampmann is one of the co-developers of a modified CRISPR interference platform that addressed the problem of CRISPR-related toxicity in stem cells. CRISPR interference technology uses a “catalytically dead” Cas9 enzyme to effectively halt gene transcription, but without cutting the DNA. As a result, and unlike CRISPR-Cas9 technology, CRISPRi shouldn’t be toxic to iPSCs or stem cell-derived neurons, Kampmann predicted.
Using the CRISPRi platform the researchers carried out screens on iPSC-derived neurons to find genes that may cause or contribute to brain diseases. They set up three complementary genetic screens based on neuronal survival, single cell RNA sequencing (scRNA-seq), and neuronal morphology. The screens generated some unexpected as well as expected findings. They identified genes that specifically extend the lifespan of neurons, but which have no comparable effect on iPSCs or on cancer cells. The screens also found genes that changed neuronal morphology, including increasing the number of neurites, which are the projections that grow from neurons and transmit nerve signals, and impacting on how frequently the neurites branched. “… our arrayed screening platform uncovered gene-specific effects on longitudinal survival and neuronal morphology,” the team stated.
Surprisingly, it was found that some typical housekeeping genes that are known to be essential for cell survival—and which had been thought to carry out the same function in all cells—in fact behave differently in neurons and in stem cells. When the researchers blocked the same housekeeping genes in these two cell types, the cells responded by activating, or inactivating, very different sets of genes. “Interestingly, we also found examples of genes that were essential in both neurons and iPSCs yet caused substantially different transcriptomic phenotypes when knocked down,” the investigators wrote. These results suggest that, in contrast with existing reasoning, housekeeping genes may not work the same way in every cell types. “They further support the idea that it is critically important to study gene function in relevant cell types, even for widely expressed genes,” the investigators stressed.
They suggest that CRISPRi is particularly well suited to studying gene function in iPSC-derived neurons. CRISPRi doesn’t cause DNA damage, it is inducible and reversible, and it partially knocks down target genes, rather than completely deleting them, which allows the functional characterization of essential genes.
Kampmann is currently using the technology to study different types of neurons with a view to understanding why some neurological diseases affect just a selective subset of neurons. In amyotrophic lateral sclerosis (ALS), for example, it’s the motor neurons that are selectively damaged.
Kampmann is also using the CRISPRi approach to study other types of brain cells, including astrocytes and microglia, which have more recently been generated using human iPSC technology. The goal is to combine CRISPRi and iPSCs into a tool that will uncover the pathways and mechanisms that underpin neurological diseases, and so help to direct the development of new therapeutic approaches. “One of the big challenges facing the field is that, for most of these disorders, the precise molecular pathways that we should target for drug development remain unclear,” said Michael Ward, MD, PhD, a physician-scientist at the NIH and co-senior author of the study in Neuron.
“With this technology, we can take skin or blood cells from a patient with a neurodegenerative disease like Alzheimer’s, turn them into neurons or other brain cells, and figure out which genes control the cellular defects associated with the disease,” said Kampmann. “The information may allow us to identify effective therapeutic targets.”
The hope is that the technology can also be used to study a range of differentiated cell types. As the authors concluded, “… our technology is not limited to neurons and should provide a paradigm for investigating specific biology of numerous other types of differentiated cells … Parallel genetic screens across the full gamut of human cell types cells may systematically uncover context-specific roles of human genes, leading to a deeper mechanistic understanding of how they control human biology and disease.”