Simultaneous CRISPR screening and spatial transcriptomics reveals intracellular, intercellular, and functional transcriptional circuits

Abstract

Pooled optical screens have enabled the study of cellular interactions, morphology, or dynamics at massive scale, but have not yet leveraged the power of highly-plexed single-cell resolved transcriptomic readouts to inform molecular pathways. Here, we present Perturb-FISH, which bridges these approaches by combining imaging spatial transcriptomics with parallel optical detection ofin situamplified guide RNAs. We show that Perturb-FISH recovers intracellular effects that are consistent with Perturb-seq results in a screen of lipopolysaccharide response in cultured monocytes, and uncover new intercellular and density-dependent regulation of the innate immune response. We further pair Perturb-FISH with a functional readout in a screen of autism spectrum disorder risk genes, showing common calcium activity phenotypes in induced pluripotent stem cell derived astrocytes and their associated genetic interactions and dysregulated molecular pathways. Perturb-FISH is thus a generally applicable method for studying the genetic and molecular associations of spatial and functional biology at single-cell resolution.

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

“…In order to achieve both high CRISPR and IVT efficiencies, we designed a chimeric promoter, where the T7 sequence is embedded within the U6 promoter as close as possible to the sgRNA without disrupting CRISPR activity. The published U6/ T7 promoter design 31,32 resulted in substantial decreases in editing efficiency ( Fig. S1c,d ), necessitating the design of a new chimeric promoter to support highly efficient screens.…”

“…FISH-based detection of sgRNAs and barcodes has recently emerged as an alternative to ISS for reading out multiplex screens 12,32,54 . These methods generally either directly detect an sgRNA with a FISH probe or detect an expanded FISH barcode often proximal to the sgRNA of interest.…”

Autism genes converge on microtubule biology and RNA-binding proteins during excitatory neurogenesis

“…Several more studies have examined ASD risk genes in parallelized in vitro screens, similarly identifying neurogenesis-related phenotypes without clear molecular correlates. Additionally, the in vitro studies chose genes based on manual curation [27][28][29][30][31] , and sometimes even further restricted to those with similar putative biological functions 27,29 . Although manually-curated gene lists tend to be more comprehensive, they also tend to introduce a degree of bias that is largely avoided by focusing on genes identified by exome-and genome-wide approaches (e.g.…”

“…They also highlighted several molecular processes as possible points of convergence, though specific processes were not consistently identified across more than one study and the pre-selection of genes reduces the generalizability of these findings. In addition to neural cells, a recent study screened perturbations of 127 manually curated ASD risk genes for disruption of ATP-mediated calcium activity in iPSC-derived astrocytes 31 . Although this study observed functional convergence in that a subset of genes resulted in dysfunctional calcium activity, specific points of molecular convergence were not characterized and the broader relevance of these findings to ASD is unclear.…”

The Current Situation and Development Prospect of Whole-Genome Screening

“…In 2019, Feldman et al developed an optical pooled screen that can systematically analyze genetic components and conduct phenotypic analysis with a wide range of spatial and temporal definitions after gene perturbation [ 133 ]. The development of an optical pool screen that can observe dynamic phenotypic changes in cells is exciting [ 134 ].…”