An In Vivo CRISPR Technique Called AAV-Perturb-seq Takes Genetic Screens to the Next Level

An In Vivo CRISPR Technique Called AAV-Perturb-seq Takes Genetic Screens to the Next Level

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Clustered regularly interspaced short palindromic repeats (CRISPR) has revolutionized research in molecular biology by enabling precise gene editing in vitro and in vivo. Gene editing in CRISPR is mediated by the Cas9 enzyme, or its derivatives, which recognize an RNA guide bound to its complement in genomic DNA with an adjacent protospacer adjacent motif (PAM) site. Upon recognition, the Cas9 enzyme can generate single or double strand deletions or insertions in genomic DNA. Recent developments have extended the use of CRISPR as a high throughput screening tool at a single cell level to study gene regulation in various disease models, such as cancer, immune dysregulation and neurodevelopment. However, these techniques are often restricted to in vitro applications, with in vivo research being limited to specific murine tissues, and lower rates of lentiviral infection.

In a recent publication in nature, Santinha et al (2023) introduce adeno-associated virus (AAV)-mediated direct in vivo single-cell CRISPR screening (AAV-Perturb-seq) to broaden the use of single cell CRISPR screening tools to in vivo functional genomics.


AAV-Perturb-seq uses adeno-associated viruses (AAV) for the efficient delivery of RNA guides to Cas9 expression cells in vivo. It is a virus mediated method to do in vivo screening of single cells after gene-based perturbations.  It’s kind of like having a genetic control panel to understand how genes impact traits and diseases. The use of AAV in in vivo delivery presents numerous advantages compared to traditional lentivirus-based screening methods. These benefits encompass the potential for systemic administration via intravenous injections, facilitating the precise targeting of a broad spectrum of tissues and cell types in animals of varying ages, all in a customizable and adaptable fashion. By leveraging AAV vectors to deliver guide RNAs (gRNAs) for gene knockout or regulatory elements for gene overexpression, scientists can investigate how these perturbations influence gene expression patterns at the single-cell level in vivo

Decoding 22q11.2 Deletion Syndrome

In this study, AAV-Perturb-seq was used to unravel the mechanism behind 22q11.2 deletion syndrome, a genetic disorder associated with a wide range of diseases. By applying gene editing and transcriptional inhibition techniques, the study identified three genes (Dgcr8, Dgcr14, and Gnb1l) within the 22q11.2 locus that lead to 40% of the transcription changes associated with the syndrome. These genes play critical roles in both known and previously undiscovered pathways that orchestrate critical neuronal functions within the brain. This discovery is a giant leap forward in our quest to understand the genetic basis of this horrible disease.

Implications for Genetic Interventions

What does this mean for future treatments? It signifies a major stride towards deciphering the complex interplay between genes and diseases. The dysregulation of specific genes associated with disease susceptibility, particularly those linked to RNA processing and synaptic function, holds the key to understanding and potentially intervening in genetic diseases.


As a new technique, AAV-Perturb-seq is very promising. It allows us to peer into the intricate world of geneotype to phenotype correlations with unprecedented precision. The recent study on 22q11.2 deletion syndrome genes is just the beginning. This could open doors to a better understanding of multiple genetic diseases and, ultimately, the development of targeted therapies. The future of precision medicine looks brighter than ever. Fingers crossed we see many more techniques like AAV Perturb-seq to come in the future.

Outsourcing Bioinformatics Analysis: How Bridge Informatics Can Help

Groundbreaking studies like these are made possible by technological advances making biological data generation, storage and analysis faster and more accessible than ever before. From pipeline development and software engineering to deploying existing bioinformatics tools, Bridge Informatics can help you on every step of your research journey.

As experts across data types from leading sequencing platforms, we can help you tackle the challenging computational tasks of storing, analyzing and interpreting genomic and transcriptomic data. Bridge Informatics’ bioinformaticians are trained bench biologists, so they understand the biological questions driving your computational analysis. Click here to schedule a free introductory call with a member of our team.

Haider M. Hassan, Data Scientist, Bridge Informatics

Haider is one of our premier data scientists. He provides bioinformatic services to clients, including high throughput sequencing, data pre-processing, analysis, and custom pipeline development. Drawing on his rich experience with a variety of high-throughput sequencing technologies, Haider analyzes transcriptional (spatial and single-cell), epigenetic, and genetic landscapes.

Before joining Bridge Informatics, Haider was a Postdoctoral Associate at the London Regional Cancer Centre in Ontario, Canada. During his postdoc, he investigated the epigenetics of late-onset liver cancer using murine and human models. Haider holds a Ph.D. in biochemistry from Western University, where he studied the molecular mechanisms behind oncogenesis. Haider still lives in Ontario and enjoys spending his spare time visiting local parks. If you’re interested in reaching out, please email [email protected] or [email protected]

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