Massively parallel in vivo Perturb-seq screening.

Area of Science:

• Genomics
• Molecular Biology
• Systems Biology

Background:

• Genomic studies have identified numerous disease risk genes, but their functions and mechanisms remain largely unknown.
• Understanding gene function in specific cell types within intact tissues is crucial for advancing disease research.
• A gap exists in methods for dissecting gene function and cell-type specificity in vivo.

Purpose of the Study:

• To describe a detailed protocol for performing massively parallel in vivo Perturb-seq.
• To provide guidance on designing and executing in vivo Perturb-seq experiments for functional genomics.
• To facilitate the adoption of in vivo Perturb-seq for diverse biological questions.

Main Methods:

• Utilized state-of-the-art gene editing tools for programmable gene perturbations.
• Employed high-content, high-resolution single-cell genomic assays for phenotypic readouts.
• Developed a protocol using versatile adeno-associated virus (AAV) vectors for in vivo delivery.

Main Results:

• Detailed step-by-step procedures from perturbation library design to AAV production and administration.
• Highlighted critical quality control checkpoints for experimental success.
• Discussed data analysis options and available software for in vivo Perturb-seq.

Conclusions:

• In vivo Perturb-seq offers a powerful approach to accelerate functional genomics studies in mammalian systems.
• This protocol empowers researchers to investigate gene function and cell-specific roles in vivo.
• The method has the potential to answer a broad array of biological questions, advancing our understanding of health and disease.