Summary: Scientists have developed a groundbreaking CRISPR screening method called in vivo Perturb-seq. This innovative technique allows for rapid and scalable analysis of how genetic changes affect individual brain cells, offering new insights into the cellular drivers of neurological diseases.

By understanding how specific cell types respond to genetic perturbations, researchers can identify potential therapeutic targets and develop more effective treatments.

Key Facts:

• A new CRISPR screen method enables rapid analysis of gene function in individual brain cells.
• This technique can be used to identify cell types susceptible to disease-causing genetic mutations.
• The scalable method allows for profiling of tens of thousands of cells in a single experiment.

Source: Scripps Research Institute

The brain is often referred to as a “black box”—one that’s difficult to peer inside and determine what’s happening at any given moment. This is part of the reason why it’s difficult to understand the complex interplay of molecules, cells and genes that underly neurological disorders.

But a new CRISPR screen method developed at Scripps Research has the potential to uncover new therapeutic targets and treatments for these conditions.

The method, outlined in a study published in Cell on May 20, 2024, provides a way to rapidly examine the brain cell types linked to key developmental genes at a scale never done before—helping unravel the genetic and cellular drivers of different neurological diseases.

“We know that certain genetic variations in our genome can make us vulnerable or resilient towards different diseases, but which specific cell types are behind a disease? Which brain regions are susceptible to the genome mutations in those cells? These are the kinds of questions we’re trying to answer,” says senior author Xin Jin, PhD, an assistant professor in the Department of Neuroscience at Scripps Research.

“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 says.

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 says.

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 says 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.

Funding:

This work and the researchers involved were supported by funding from the Dorris Scholar Award, the Frank J. Dixon Fellowship, the Mark Pearson Endowed Fellowship, the Stanley Center for Psychiatric Research at the Broad Institute of MIT and Harvard, the Simons Foundation Autism Research Initiative (grant SFARI #736613), the National Institute of Health (grant R01HG012819), the Impetus grant, the One Mind Rising Star Award, the Klingenstein-Simons Fellowship, the Mathers Foundation, the Baxter Young Investigator Award, the Larry L. Hillblom Foundation, the Scripps Collaborative Innovative Fund, the Chan Zuckerberg Initiative, the Conrad Prebys Foundation, the Astera Institute, and the James Fickle.

About this CRISPR and neuroscience research

Author: Press Office
Source: Scripps Research Institute
Contact: Press Office – Scripps Research Institute
Image: The image is credited to Neuroscience News

Original Research: Open access.
“Massively parallel in vivo Perturb-seq reveals cell-type-specific transcriptional networks in cortical development” by Xin Jin et al. Cell

Abstract

Massively parallel in vivo Perturb-seq reveals cell-type-specific transcriptional networks in cortical development

Highlights

• Fast-onset AAVs reach high expression within two days in vivo
• Transposon enhances expression and perturbation in brain and peripheral nervous systems
• Cell-type-restricted effects of Foxg1, whose perturbation leads to hybrid cell states
• Modular platform for in vivo phenotypic CRISPR screen with scale

Summary

Leveraging AAVs’ versatile tropism and labeling capacity, we expanded the scale of in vivo CRISPR screening with single-cell transcriptomic phenotyping across embryonic to adult brains and peripheral nervous systems.

Through extensive tests of 86 vectors across AAV serotypes combined with a transposon system, we substantially amplified labeling efficacy and accelerated in vivo gene delivery from weeks to days.

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.

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.

Compatible with various phenotypic measurements (single-cell or spatial multi-omics), it presents a flexible approach to interrogate gene function across cell types in vivo, translating gene variants to their causal function.