Stitch-seq: Scalable CRISPR gene expression response profiling.

Area of Science:

• Genomics
• Molecular Biology
• Biotechnology

Background:

• Single-cell profiling of genetic perturbations is crucial for understanding gene-phenotype links.
• Current methods are costly and complex, limiting systematic studies of cellular programs.

Purpose of the Study:

• To introduce Stitch-seq, a high-throughput pooled functional genomics sequencing method.
• To enable simultaneous capture of CRISPR perturbations and targeted gene/protein expression across millions of cells.
• To provide an accessible modality for dissecting complex biological pathways.

Main Methods:

• Stitch-seq uses single-cell droplet-based overlap-extension reverse-transcription PCR.
• It physically links gene expression to perturbation identifiers without cell barcoding or extensive sequencing.
• Validated using simplified models and benchmarked against single-cell RNA-sequencing in an EMT model.

Main Results:

• Stitch-seq demonstrated high fidelity in simplified models.
• Multi-omic Stitch-seq was benchmarked against single-cell RNA-sequencing.
• Applied to map transcriptional responses during TGF-β-induced EMT in MCF10A cells across five time points.

Conclusions:

• Stitch-seq is a powerful and accessible method for large-scale multi-omic gene expression readouts.
• It facilitates the routine dissection of complex biological pathways.
• Enables efficient interrogation of dynamic cellular programs through genetic perturbations.