Information collected with the group’s CrAAVe-seq platform. Credit score: Indigo Rose, Kampmann lab, UCSF.
Over the previous few a long time, biomedical researchers and neuroscientists have devised more and more superior strategies to review and alter neurophysiological processes. These embrace CRISPR (Clustered Often Interspaced Quick Palindromic Repeats), a classy device to edit particular genes in some animals, together with mice, rats, zebrafish and fruit flies.
Researchers at College of California, San Francisco led by Martin Kampmann just lately launched a extra exact CRISPR screening platform that may be utilized straight in residing tissue, enabling the screening of a bigger variety of genes without delay. The brand new method, known as CRISPR screening by AAV episome sequencing (CrAAVe-seq), was launched in a paper revealed in Nature Neuroscience.
“Human cell-based systems are valuable but cannot fully capture the complexity of the brain,” Biswa Ramani, co-first writer of the paper, instructed Medical Xpress. “Mice often remain the most effective model for many neurological diseases because their brains preserve the diversity and organization of cell types that cannot be replicated in a dish.”
Ramani and his colleagues within the Kampmann lab have been on the lookout for a device that may enable them to quickly display a whole lot and even 1000’s of genetic modifiers straight within the mouse mind, with out requiring extremely costly processes or tools. Such a platform might be used to conduct large-scale and systematic research involving mice which might be genetically engineered to imitate particular elements of human neurological issues.
“We were inspired by thinking about out-of-the-box ideas to address the limitations of CRISPR screens done in cells grown in a petri dish,” mentioned Indigo Rose, co-first writer of the paper. “Both Biswa and I wished there was a tool where we could do CRISPR screens directly in the brain. After researching available approaches, we realized we would have to build it ourselves.”
Information collected with the group’s CrAAVe-seq platform. Credit score: Indigo Rose, Kampmann lab, UCSF.
The primary goal of this latest research was thus to plot an experimental platform that helps the screening of 1000’s of genes throughout totally different areas of the mind in parallel, whereas additionally successfully concentrating on particular cell populations. The group wished this system to even be relevant in vivo (i.e., on dwell animals).
“Our CRISPR screening platform leverages adeno-associated virus (AAV) to deliver sgRNAs into the intact mouse brain, as well as the ability to separate and isolate the remaining AAV genomes from the brain after a period of time,” defined Ramani.
“We then pair this with genetic tools that allow us to selectively ‘capture’ and sequence only the sgRNAs that were present in a specific cell type of interest.”
By combining these two distinct approaches, Ramani, Rose and their colleagues have been in a position to research the results of perturbing 1000’s of genes throughout hundreds of thousands of cells within the mouse mind in vivo. Notably, their method additionally allowed them to focus their screening on particular cell populations, comparable to neurons, astrocytes or specialised neuron subtypes.
“CrAAVe-seq delivers specially designed sgRNAs into a live mouse brain where they disrupt a specific gene, usually a different one in different neighboring cells,” mentioned Rose.
“We do this simultaneously with many thousands of different genes. Our study uncovered the scalability and reproducibility of in vivo CRISPR screens using CrAAVe-seq, enabling high-throughput analysis of large gene libraries and precise investigation of specific brain regions or cell populations.”
In comparison with typical CRISPR strategies, the method devised by these researchers leverages sgRNAs, or single-guide RNA. These are engineered RNA molecules that ‘file’ their presence in a selected sort of cell by altering a part of their sequence. These alterations are what finally permits researchers to exactly goal their CRISPR screens to cowl particular cell populations.
Information collected with the group’s CrAAVe-seq platform. Credit score: Indigo Rose, Kampmann lab, UCSF.
“We were most impressed by how robustly and consistently neuron-essential genes emerged from our large-scale sgRNA library, even when tested in a single mouse,” mentioned Ramani.
“This underscores the scalability of our approach, reaching orders of magnitude beyond what was previously possible. By extending the method across multiple mice, we can further boost reproducibility.”
The scalable genetic screening platform devised by this group of researchers might quickly pave the best way for brand new research specializing in a variety of genetic and neural processes. Sooner or later, it might assist to uncover particular mobile and molecular processes that contribute to the event of particular ailments or neuropsychiatric issues.
“Our study establishes a foundation by identifying baseline essential neuronal genes, positioning us to next define genes that become essential under stress conditions, including models of neurodegeneration and in selectively vulnerable neuronal populations,” mentioned Ramani. “We are now continuing to refine the platform to extend its utility to other phenotypes.”
“We also plan to apply CrAAVe-seq to other cell populations, as well as performing it in mouse models of neurological disease,” added Rose.
Written for you by our writer Ingrid Fadelli, edited by Sadie Harley, and fact-checked and reviewed by Robert Egan—this text is the results of cautious human work. We depend on readers such as you to maintain unbiased science journalism alive.
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Extra info:
Biswarathan Ramani et al, CRISPR screening by AAV episome-sequencing (CrAAVe-seq): a scalable cell-type-specific in vivo platform uncovers neuronal important genes, Nature Neuroscience (2025). DOI: 10.1038/s41593-025-02043-9.
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