In vivo multiomic Perturb-seq with enhanced nuclear gRNA capture

Area of Science:

• Genomics and Molecular Biology
• Neuroscience
• Epigenetics

Background:

• * In vivo CRISPR screening enables studying gene function in complex biological systems.
• * Current methods struggle with efficient recovery of guide RNAs (gRNAs) from cell nuclei for multiomic analysis.
• * This limitation hinders high-resolution mapping of transcriptomic and epigenomic changes induced by perturbations.

Purpose of the Study:

• * To develop an improved in vivo CRISPR screening platform for high-fidelity, high-recovery gRNA assignment.
• * To enable scalable, perturbation-resolved single-nucleus multiomics.
• * To investigate cell-type-specific transcriptomic and epigenomic phenotypes of neurodevelopmental disorder risk genes.

Main Methods:

• * Developed 'in vivo multiomic Perturb-seq', a novel platform combining nuclear transcript anchoring with gRNA-specific capture and amplification.
• * Applied the platform to the developing cortex to study neurodevelopmental disorder risk genes.
• * Performed single-nucleus multiomic profiling to capture transcriptomic and epigenomic data.

Main Results:

• * Achieved high-fidelity and high-recovery gRNA assignment from nuclei.
• * Enabled scalable single-nucleus multiomics with precise perturbation resolution.
• * Identified cell-type-specific transcriptomic and epigenomic perturbation phenotypes associated with neurodevelopmental disorder risk genes.

Conclusions:

• * The developed platform overcomes previous limitations in in vivo CRISPR screening.
• * It facilitates comprehensive analysis of gene perturbations at single-cell resolution in the brain.
• * This advancement aids in understanding the molecular mechanisms underlying neurodevelopmental disorders.