Connecting guides, genes, and morphology to expand optical pooled screening

Functional genomics aims to connect gene perturbations with their phenotypic effects, but most current screening methods can only capture part of the picture.

Optical pooled screening (OPS) offers image-based insights, while approaches like Perturb-seq profile the transcriptome. What’s missing among these approaches is a way to integrate these data types in a single, scalable experiment.

In our latest bioRxiv preprint, we introduce Direct In Sample Sequencing (DISS), a chemistry that directly sequences native RNA within intact, fixed cells. By combining in situ guide RNA sequencing with transcriptomic, protein detection, and cellular morphology, DISS delivers deeper, more informative screens.

A new approach to in situ RNA sequencing

DISS sequences guide RNAs using single-sided probes that hybridize to a known CRISPR guide RNA scaffold sequence, then extend to capture downstream RNA.

Key features of DISS:

• Hybridizes directly to unmodified sgRNAs.

• Removes the need for engineered constructs or dual-flank probe designs.

• Eliminates reliance on large probe panels.

• Enables robust detection of guide RNAs in their native context.

Demonstrating DISS in CRISPR-perturbed lung cancer cells

To validate the platform, our team performed a pooled CRISPR screen targeting Wnt, inflammation, and TGF signaling pathways in A549 lung cancer cells. By combining DISS-based 3’ transcriptome profiling and sgRNA sequencing with protein and morphological readouts, we captured dynamic cellular responses over multiple time points in a single experiment.

For example, when cells with an IL1R1 (receptor for interleukin-1β) knockout were treated with IL-1β, both protein and RNA measurements showed a collapse of canonical inflammatory signaling. Within 30 minutes of treatment, we observed protein-level changes such as loss of p38 and HSP27 phosphorylation, while RNA readouts confirmed broad suppression of NF-κB–associated genes like CXCL8, CCL2, and NFKBIA.

The data also revealed crosstalk between IL-1 and TGF pathways, with IL-1β triggering Smad2 phosphorylation independently of TGFBR2 signaling, a phenomenon previously observed that highlights the platform’s strength in linking genetic perturbations to complex phenotypes.

Morphological insights into cellular remodeling

Beyond acute signaling events, molecular and morphological profiling revealed how TGFBR2 knockout reshaped cytoskeletal organization.

This was supported by:

• Decreased expression of genes associated with cytoskeletal regulation
• Reduced adhesion and stress response signatures
• Concordant protein changes reinforcing the structural remodeling

Together, these findings show how DISS connects molecular perturbations to changes in cell structure, signaling, and transcription within a single experimental framework.

A foundation for next-generation functional genomics

By integrating RNA sequencing, protein quantification, and cell morphology in an automated OPS workflow, AVITI24 provides a new lens for understanding how genetic changes propagate through multiple layers of cellular biology.

With Teton Atlas and AVITI24, CRISPR becomes clarity so you can move from pooled screens to prioritized targets, without the noise.

Dive into the data

Explore the complete methodology, results, and figures in our bioRxiv preprint: Direct In-Sample Sequencing of the 3′ Transcriptome Expands the Capabilities of Optical Pooled Screens