A Pooled Cell Painting CRISPR Screening Platform Enables de novo Inference of Gene Function by Self-supervised Deep Learning

Pooled CRISPR screening has emerged as a powerful method of mapping gene functions thanks to its scalability, affordability, and robustness against well or plate-specific confounders present in array-based screening [1][1]–[6][2]. Most pooled CRISPR screens assay for low dimensional phenotypes (e.g. fitness, fluorescent markers). Higher-dimensional assays such as perturb-seq are available but costly and only applicable to transcriptomics readouts [7][3]–[11][4]. Recently, pooled optical screening, which combines pooled CRISPR screening and microscopy-based assays, has been demonstrated in the studies of the NFkB pathway, essential human genes, cytoskeletal organization and antiviral response [12][5]–[15][6]. While the pooled optical screening methodology is scalable and information-rich, the applications thus far employ hypothesis-specific assays. Here, we enable hypothesis-free reverse genetic screening for generic morphological phenotypes by re-engineering the Cell Painting [16][7] technique to provide compatibility with pooled optical screening. We validated this technique using well-defined morphological genesets (124 genes), compared classical image analysis and self-supervised learning methods using a mechanism-of-action (MoA) library (300 genes), and performed discovery screening with a druggable genome library (1640 genes) [17][8]. Across these three experiments we show that the combination of rich morphological data and deep learning allows gene networks to emerge without the need for target-specific biomarkers, leading to better discovery of gene functions. ### Competing Interest Statement The authors are current (S.S., B.L., M.M.S., N.R., A.E., Z.F.P., J.W.B., D.K., E.S., A.K., M.R.S., and C.C.) or former (E.L., P.S., J.E.M., A.K., J.C., F.R., and D.F.) employees and shareholders of insitro inc. [1]: #ref-1 [2]: #ref-6 [3]: #ref-7 [4]: #ref-11 [5]: #ref-12 [6]: #ref-15 [7]: #ref-16 [8]: #ref-17