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

Abstract

Optical pooled screening (OPS) is a highly scalable method for linking image-based phenotypes with cellular perturbations. However, it has thus far been restricted to relatively low-plex phenotypic readouts in cancer cell lines in culture, due to limitations associated within situsequencing (ISS) of perturbation barcodes. Here, we developed PerturbView, an OPS technology that leveragesin vitrotranscription (IVT) to amplify barcodes prior to ISS, enabling screens with highly multiplexed phenotypic readouts across diverse systems, including primary cells and tissues. We demonstrate PerturbView in iPSC-derived neurons, primary immune cells, and tumor tissue sections from animal models. In a screen of immune signaling pathways in primary bone marrow-derived macrophages, PerturbView uncovered both known and novel regulators of NFκB signaling. Furthermore, we combined PerturbView with spatial transcriptomics in tissue sections from a mouse xenograft model, paving the way toin vivoscreens with rich optical and transcriptomic phenotypes. PerturbView broadens the scope of OPS to a wide range of models and applications.

CROPseq-multi: a versatile solution for multiplexed perturbation and decoding in pooled CRISPR screens

“…Recently developed approaches for in situ genotyping with in vitro transcription provide an alternative strategy to mRNA-detection for optical pooled screens 53,54 . A similar strategy may be compatible with a T7-promoter-modified CROPseq-multi approach for compatibility with both mRNA-based and in vitro transcription-based detection.…”

Integrated combinatorial functional genomics and spatial transcriptomics of tumors decodes genotype to phenotype relationships

“…Moreover, our currently employed perturbation plasmids (Extended Data Fig. 3) are expected to be directly compatible with orthogonal barcode mapping strategies 13,20,56,57 and only minor adjustments to match the interrogated sequences are required to use CHOCOLATE-G2P in conjunction with imaging-based platforms 58,59 . Since multiple spatial omics technologies hinge on the availability of robust RNA-detection probes, a requirement not always satisfied as evidenced by the inefficacy of PERTURB-CAST to detect 5/38 barcodes tested in this study, prospective massively parallel assays 54 based on CHOCOLAT-G2P could be leveraged to screen for reliable probes.…”

“…As CHOCOLATE-G2P may be extended to other alterations 57,60,61 , disease models 3,7,12,52,62 and spatial omics technologies 5,13,59 , we envisage a broad range of applications to finally decode the relationships between complex genotypes and phenotypes.…”

Transcriptome-wide characterization of genetic perturbations

“…Average expression is a widely used and highly interpretable readout, but the transcriptional state of individual cells may vary in ways that are poorly captured in pseudo-bulk (for example, due to the presence of multiple cell types) and are better understood with modeling of cell type variability (Lopez et al 2018). In addition, some biological processes are better assayed using alternative modalities, including mRNA splicing, chromatin state, protein level, and imaging, all of which are now being studied at scale with single cell CRISPR screens (Rubin et al 2019; Feldman et al 2019; Gu et al 2023; Kudo et al 2023; Binan et al 2023; Xu et al 2023) We predict that future methods building on our approach will have broad application to these other phenotypic readouts as well as to the study of non-genetic perturbations such as drugs and development.…”