A new CRISPR screen method developed at Scripps Research has the potential to improve studies into the genetic underpinnings of human diseases and disorders. The method was outlined in Cell, in a paper titled, “Massively parallel in vivo Perturb-seq reveals cell-type-specific transcriptional networks in cortical development.”
The paper’s authors, led by Xin Jin, PhD, a neuroscientist at Scripps Research, summarized the limitations of existing CRISPR screens: 1) They have difficulty accomplishing the scalable labeling and perturbation of sufficient numbers of cells in vivo. 2) They have difficulty deconvoluting each cell’s perturbation identity in the sparse single-cell omics data. 3) They rely on lentiviral vectors, which are known to have limited in vivo penetration and thermostability, hampering systemic screens in hard-to-reach tissues such as the central and peripheral nervous systems.
To address these issues, Jin and colleagues developed an AAV-based, massively parallel in vivo Perturb-seq platform to target broad tissues and cell types with gene-expression-based characterization at single-cell resolution. To demonstrate the platform, the scientists used it to study the development of embryonic brains in mice.
“Our proof-of-principle in utero screen identified the pleiotropic effects of Foxg1, highlighting its tight regulation of distinct networks essential for cell fate specification of Layer 6 corticothalamic neurons,” the article’s authors wrote. “Notably, our platform can label >6% of cerebral cells, surpassing the current state-of-the-art efficacy at <0.1% by lentivirus, to achieve analysis of over 30,000 cells in one experiment and enable massively parallel in vivo Perturb-seq.”
“With this new technology, we want to build a more dynamic picture across brain region, across cell type, across the timing of disease development, and really start understanding how the disease happened—and how to design interventions.”
Thanks to over a decade’s efforts in human genetics, scientists have had access to long lists of genetic changes that contribute to a range of human illnesses, but knowing how a gene causes a disease is very different than knowing how to treat the illness itself. Every risk gene may impact one or several different cell types. Comprehending how those cell types—and even individual cells—impact a gene and affect disease progression is key to understanding how to ultimately treat that disease.
This is why Jin, along with the study’s first author, Xinhe Zheng, a PhD candidate and the Frank J. Dixon Graduate Fellow at Scripps Research, co-invented the new technique, named in vivo Perturb-seq. This method leverages CRISPR-Cas9 technology and a readout, single-cell transcriptomic analysis, to measure its impact on a cell: one cell at a time. Using CRISPR-Cas9, scientists can make precise changes to the genome during brain development, and then closely study how those changes affect individual cells using single-cell transcriptomic analysis—for tens of thousands of cells in parallel.
“Our new system can measure individual cells’ response after genetic perturbations, meaning that we can paint a picture of whether certain cell types are more susceptible than others and react differently when a particular mutation happens,” Jin said.
Previously, the method for introducing the genetic perturbations into the brain tissue was very slow, often taking days or even weeks, which created suboptimal conditions for studying gene functions related to neurodevelopment. But Jin’s new screening method allows for rapid expression of perturbation agents in living cells within 48 hours—meaning scientists can quickly see how specific genes function in different types of cells in a very short amount of time.
The method also enables a level of scalability that was previously impossible—the research team was able to profile more than 30,000 cells in just one experiment, 10–20 times accelerated from the traditional approaches. In many of the brain regions they examined, such as the cerebellum, they were able to collect tens of thousands of cells that previous labeling methods could not reach.
In a pilot study using this new technology, Jin and her team’s interest was piqued when they saw a genetic perturbation elicit different effects when perturbed in different cell types. This is important because those impacted cell types are the sites of action for particular diseases or genetic variants. “Despite their smaller population representations, some low-abundant cell types may have a stronger impact than others by the genetic perturbation, and when we systematically look at other cell types across multiple genes, we see patterns. That’s why single-cell resolution—being able to study every cell and how each one behaves—can offer us a systematic view,” Jin said.
With her new technology in hand, Jin plans to apply it to better understand neuropsychiatric conditions and how certain cell types correspond with various brain regions. Moving forward, Jin said she’s excited to see this type of technology applied to additional cell types in other organs in the body to better understand a wide range of diseases in terms of tissue, development, and aging.
