Pooled CRISPR screening with single-cell transcriptome readout.

Area of Science:

• Genomics
• Molecular Biology
• Bioinformatics

Background:

• CRISPR genetic screens accelerate biological discovery but face limitations.
• Pooled screens offer high throughput but limited readouts (e.g., proliferation).
• Arrayed screens provide comprehensive readouts (e.g., transcriptome) but are low throughput.

Purpose of the Study:

• To develop a broadly applicable workflow combining pooled CRISPR screening with single-cell RNA sequencing.
• To directly link guide RNA expression to transcriptome responses at the single-cell level.
• To enable high-throughput functional dissection of complex regulatory mechanisms and heterogeneous cell populations.

Main Methods:

• Developed CRISPR droplet sequencing (CROP-seq).
• Integrated pooled CRISPR screening with single-cell RNA sequencing.
• Enabled direct linkage of guide RNA expression to transcriptome responses in thousands of individual cells.

Main Results:

• CROP-seq provides single-cell transcriptome resolution for pooled CRISPR screens.
• The workflow facilitates high-throughput functional dissection.
• Enables analysis of complex regulatory mechanisms and heterogeneous cell populations.

Conclusions:

• CROP-seq overcomes limitations of existing CRISPR screening methods.
• This method advances functional genomics by enabling comprehensive molecular readouts at high throughput.
• Facilitates deeper understanding of gene function and cellular responses.