Summary: Scientists have developed a revolutionary CRISPR screening method called Perturb-seq in vivo. This innovative technique enables rapid and scalable analysis of how genetic changes affect individual brain cells, providing new insights into the cellular drivers of neurological diseases.
By understanding how specific cell types respond to genetic disruptions, researchers can identify potential therapeutic targets and develop more effective treatments.
Highlights:
- A new CRISPR screening 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 tens of thousands of cells to be profiled in a single experiment.
Source: Scripps Research Institute
The brain is often referred to as a “black box,” a box that is difficult to look inside and determine what is happening at any given moment. This is part of the reason why it is difficult to understand the complex interplay of molecules, cells and genes that cause neurological disorders.
But a new CRISPR screening method developed at Scripps Research has the potential to discover new therapeutic targets and treatments for these conditions.
The method, described in a study published in Cell on May 20, 2024, provides a way to rapidly examine brain cell types linked to key developmental genes on a scale never before done, helping to elucidate the genetic and cellular drivers of different neurological diseases.
“We know that certain genetic variations in our genome can make us vulnerable or resilient to different diseases, but which specific cell types cause a disease? Which brain regions are sensitive to genome mutations in these cells? These are the kinds of questions we are trying to answer,” says lead author Xin Jin, PhD, assistant professor in the Department of Neuroscience at Scripps Research.
“With this new technology, we want to paint a more dynamic picture of brain regions, cell types and timing of disease development, and really begin to understand how the disease occurred and how to design interventions. »
Through more than a decade of effort in human genetics, scientists have gained access to long lists of genetic changes that contribute to a range of human diseases, but knowing how a gene causes a disease is very different from knowing how treat the disease itself.
Each risk gene can impact one or more different cell types. Understanding how these cell types, and even individual cells, impact a gene and affect disease progression is essential to understanding how to treat this disease.
That’s why Jin, along with the study’s first author, Xinhe Zheng, a doctoral student and Frank J. Dixon Graduate Fellow at Scripps Research, co-invented the new technique, named alive Disrupt-seq. This method uses CRISPR-Cas9 technology and single-cell transcriptomic readout 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, then closely study how these changes affect individual cells using single-cell transcriptomic analysis, for tens of thousands of cells in parallel.
“Our new system can measure the response of individual cells after genetic disruptions, which means we can determine whether certain cell types are more sensitive than others and react differently when a particular mutation occurs,” explains Jin.
Previously, the method of introducing genetic disruptions into brain tissue was very slow, often taking days or even weeks, creating suboptimal conditions for studying gene functions related to neurodevelopment.
But Jin’s new screening method allows for rapid expression of disruptive agents in living cells within 48 hours, meaning scientists can quickly see how specific genes function in different cell types in a very short 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 a single experiment, a 10- to 20-fold speedup over traditional approaches.
In many regions of the brain 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 his team’s interest was piqued when they found that a genetic disruption caused different effects when disrupted in different cell types. This is important because the cell types affected are the sites of action of particular diseases or genetic variants.
“Despite their smaller representation in the population, some low-abundance cell types can have a stronger impact than others due to genetic disruption, and when we systematically examine other cell types across multiple genes, we observe trends. This is why single-cell resolution, that is, the ability to study each cell and its behavior, can give us a systematic view,” explains Jin.
With his new technology in hand, Jin plans to apply it to better understand neuropsychiatric conditions and how certain cell types correspond to various regions of the brain.
In the future, Jin says she is excited to see this type of technology applied to other cell types in other organs of the body to better understand a wide range of diseases in terms of tissues, development and of 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. (SFARI grant #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 research CRISPR and neuroscience
Author: Press office
Source: Scripps Research Institute
Contact: Press Office – Scripps Research Institute
Picture: Image is credited to Neuroscience News
Original research: Free access.
“Massively parallel in vivo Perturb-seq reveals cell type-specific transcription networks in cortical development” by Xin Jin et al. Cell
Abstract
Massively parallel in vivo disruption-seq reveals cell type-specific transcriptional networks in cortical development
Strong points
- Fast-acting AAVs reach high expression within two days alive
- Transposon enhances expression and disruption in brain and peripheral nervous systems
- Effects restricted to cell type Fox1whose disruption leads to hybrid cellular states
- Modular platform for alive Phenotypic CRISPR screen with ladder
Summary
By leveraging the versatile tropism and labeling capacity of AAVs, we have expanded the scale of alive CRISPR screening with single-cell transcriptomic phenotyping in embryonic to adult brains and peripheral nervous systems.
Through extensive testing of 86 vectors on AAV serotypes combined with a transposon system, we significantly amplified the marking efficiency and accelerated alive delivery of genes from a few weeks to a few days.
Our proof of principle in utero The screen identified the pleiotropic effects of Fox1highlighting its tight regulation of distinct networks essential for cell fate specification of layer 6 corticothalamic neurons.
Notably, our platform can label >6% of brain cells, exceeding the current peak efficiency of <0.1% by lentiviruses, to achieve analysis of more than 30,000 cells in a single experiment and enable massive analysis. parallel. alive Disrupt-seq.
Compatible with various phenotypic measurements (single-cell or spatial multi-omics), it presents a flexible approach to interrogate gene function across all cell types alivetranslating genetic variants into their causal function.