Pooled CRISPR-activation screening coupled with single-cell RNA-seq in mouse embryonic stem cells

Area of Science:

• Molecular Biology
• Genomics
• Stem Cell Biology

Background:

• CRISPR/Cas9 screens are vital for identifying biological regulators.
• Combining pooled CRISPR screening with single-cell RNA sequencing (scRNA-seq) enables comprehensive, large-scale analysis of individual perturbations.
• This approach allows unbiased interrogation of gene function and regulation at the cellular level.

Purpose of the Study:

• To present a detailed protocol for performing pooled CRISPR-activation screens.
• To utilize 10× Genomics single-cell RNA sequencing (scRNA-seq) as a readout for these screens.
• To enable unbiased, high-throughput analysis of gene function in mouse embryonic stem cells.

Main Methods:

• Development of a protocol for pooled CRISPR-activation screening.
• Application in mouse embryonic stem cells.
• Readout using 10× Genomics single-cell RNA sequencing (scRNA-seq).

Main Results:

• The protocol enables parallel assessment of individual perturbations.
• Comprehensive and scalable analysis of gene function is achieved.
• Unbiased interrogation of gene regulation at the single-cell level is facilitated.

Conclusions:

• The presented protocol effectively combines pooled CRISPR-activation screening with scRNA-seq.
• This method provides a powerful tool for dissecting gene function and regulatory networks in stem cells.
• It facilitates unbiased discovery of key regulators in biological processes.