Over the past few decades, biomedical researchers and neuroscientists have devised increasingly advanced techniques to study and alter neurophysiological processes. These include CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), a sophisticated tool to edit specific genes in some animals, including mice, rats, zebrafish and fruit flies.
Researchers at University of California, San Francisco led by Martin Kampmann recently introduced a more precise CRISPR screening platform that can be applied directly in living tissue, enabling the screening of a larger number of genes at once. The new technique, called CRISPR screening by AAV episome sequencing (CrAAVe-seq), was introduced in a paper published in Nature Neuroscience.
“Human cell-based systems are valuable but cannot fully capture the complexity of the brain,” Biswa Ramani, co-first author of the paper, told 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 in the Kampmann lab were looking for a tool that would allow them to rapidly screen hundreds or even thousands of genetic modifiers directly in the mouse brain, without requiring highly expensive processes or equipment. Such a platform could be used to conduct large-scale and systematic studies involving mice that are genetically engineered to mimic specific aspects of human neurological disorders.
“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,” said Indigo Rose, co-first author 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.”
The main objective of this recent study was thus to devise an experimental platform that supports the screening of thousands of genes across different regions of the brain in parallel, while also effectively targeting specific cell populations. The team wanted this technique to also be applicable in vivo (i.e., on live 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,” explained 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 were able to study the effects of perturbing thousands of genes across millions of cells in the mouse brain in vivo. Notably, their approach also allowed them to focus their screening on specific cell populations, such as neurons, astrocytes or specialized 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,” said 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.”
Compared to conventional CRISPR techniques, the approach devised by these researchers leverages sgRNAs, or single-guide RNA. These are engineered RNA molecules that ‘record’ their presence in a specific type of cell by altering part of their sequence. These alterations are what ultimately allows researchers to precisely target their CRISPR screens to cover specific cell populations.
“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,” said 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 team of researchers could soon pave the way for new studies focusing on a wide range of genetic and neural processes. In the future, it could help to uncover specific cellular and molecular processes that contribute to the development of specific diseases or neuropsychiatric disorders.
“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,” said 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 author Ingrid Fadelli, edited by Sadie Harley, —this article is the result of careful human work. We rely on readers like you to keep independent science journalism alive.
If this reporting matters to you,
please consider a donation (especially monthly).
You’ll get an ad-free account as a thank-you.
More information:
Biswarathan Ramani et al, CRISPR screening by AAV episome-sequencing (CrAAVe-seq): a scalable cell-type-specific in vivo platform uncovers neuronal essential genes, Nature Neuroscience (2025). DOI: 10.1038/s41593-025-02043-9.
The content is provided for information purposes only.