Direct In-Sample Sequencing of the 3' Transcriptome Expands the Capabilities of Optical Pooled Screens

Daniel J. Honigfort; Pedro Belda‐Ferre; David T. White; Kousik Sundararajan; Mariam Dawood; Jake A. LeVieux; Jacob Moreno; Xiaodong Qi; Kyle S. Metcalfe; Duuluu Naranbat; Andrew Altomare; Connor Thompson; Carlos Ruiz Perez; Bryan R. Lajoie; Hyuk-Jin Kwon; Pazinah Bhadha; Tristin Rammel; John Rabalais; Matthew William Kellinger; Semyon Kruglyak; Sinan Arslan; Michael J. R. Previte

doi:10.1101/2025.10.11.681797

GenomicsbioRxivOct 11, 2025

Preprint

Direct In-Sample Sequencing of the 3' Transcriptome Expands the Capabilities of Optical Pooled Screens

Daniel J. Honigfort iD, Pedro Belda‐Ferre iD, David T. White, Kousik Sundararajan iD, Mariam Dawood, Jake A. LeVieux, Jacob Moreno, Xiaodong Qi iD, Kyle S. Metcalfe iD, Duuluu Naranbat, Andrew Altomare, Connor Thompson iD, Carlos Ruiz Perez, Bryan R. Lajoie iD, Hyuk-Jin Kwon, Pazinah Bhadha, Tristin Rammel, John Rabalais, Matthew William Kellinger, Semyon Kruglyak iD, Sinan Arslan iD, Michael J. R. Previte iD

Abstract

We present a platform that directly sequences single guide RNAs and endogenous 3'UTRs in fixed cells while simultaneously measuring protein abundance and cellular morphology. We demonstrate platform capability by performing optical pooled screening of CRISPR-perturbed lung cancer cells. This approach unites direct in-sample RNA sequencing with complementary phenotypic readouts, enabling comprehensive, scalable, and functional genomics analyses within a single experiment.

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