Mapping variation in the morphological landscape of human cells with optical pooled CRISPRi screening

Abstract

The contributions of individual genes to cell-scale morphology and cytoskeletal organization are challenging to define due to the wide intercellular variation of these complex phenotypes. We leveraged the controlled nature of image-based pooled screening to assess the impact of CRISPRi knockdown of 366 genes on cell and nuclear morphology in human U2OS osteosarcoma cells. Screen scale-up was facilitated by a new, efficient barcode readout method that successfully genotyped 85% of cells. Phenotype analysis using a deep learning algorithm, the β-variational autoencoder, produced a feature embedding space distinct from one derived from conventional morphological profiling, but detected similar gene hits while requiring minimal design decisions. We found 45 gene hits and visualized their effect by rationally constrained sampling of cells along the direction of phenotypic shift. By relating these phenotypic shifts to each other, we construct a quantitative and interpretable space of morphological variation in human cells.

Highly multiplexed, image-based pooled screens in primary cells and tissues with PerturbView

“…To avoid inhibition of IVT by PFA fixation 25 , we added a decrosslinking step 29 , followed by sgRNA overnight amplification with T7 RNAP (Methods). Following IVT, we performed the conventional OPS protocol 21,30 and sequenced the first base of the sgRNA. Constructs containing the T7 promoter produced sgRNA signal as bright nuclear foci ( Fig.…”

“…A primer crosslinking protocol was adopted from Labitigan et al 21 for all data, except Figure 1b-c which uses the original OPS protocol 13 . An amine-modified primer was hybridized at 1 μM in PBST for 30 minutes at room temperature.…”

“…In this latter class, optical pooled screens (OPS) use targeted in situ sequencing (ISS) of perturbation barcodes to perform highly scalable imaging screens. OPS has been used to study perturbation effects on immune signaling 13 , viral infection 16 , the unfolded protein response 17 , and cell morphology [18][19][20][21] .…”

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

“…Recently, pooled optical screening methods have employed bespoke phenotypic assays designed for studying specific biological questions such as the NFkB pathway, antiviral response, cytoskeletal organization, and essential genes [12][13][14][15] . In contrast, a generic morphological assay would enable hypothesis-free biological exploration.…”

“…Higher-dimensional assays such as perturb-seq are available but costly and only applicable to transcriptomics readouts [7][8][9][10][11] . 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][13][14][15] . While the pooled optical screening methodology is scalable and information-rich, the applications thus far employ hypothesis-specific assays.…”