Background
This study demonstrates the application of Direct In Sample Sequencing (DISS) for optical pooled screening (OPS) in A549 lung epithelial cells. By integrating DISS into the OPS workflow, we directly sequenced perturbation identities within the same cells used for imaging, linking CRISPR-induced changes to phenotypic and protein-level readouts. This approach enables high-content, multiplexed analysis of how inflammation, Wnt, and TGF pathway perturbations shape cellular responses under IL-1 and TGF stimulation across a 48-hour time course.
Methods
A549 Cas9-expressing cells were transfected with a 28-guide CRISPR library targeting CTNNB1, GSK3B, IL1R1, SMAD7, and TGFBR2, along with OR1I1 (olfactory receptor; no expected phenotype in A549) and non-targeting controls. Cells were profiled at baseline and following IL-1 or TGF stimulation at 30 mins, 1 hr, 8 hrs, 24 hrs, and 48 hrs. Guide sequencing, cell paint, and protein detection was performed using the Teton Atlas Low Output workflow with the MAPK protein panel.
Results
Across the experiment, we studied approximately 228,000 single cells, providing broad coverage of each perturbation and condition. On average, we profiled 26,000 cells per targeted gene, corresponding to roughly 4,000 cells per gene per condition across the six time points. This dataset captures how CRISPR perturbations alter cytokine-driven signaling over time.