Figure 3.
Spatial CRISPR screens reveal intracellular and intercellular immune regulatory mechanisms. Building upon the Pro-Code method, Perturb-map was developed to integrate spatial information with CRISPR perturbations, uncovering how tumor clonality shapes the composition of the TME. CRISPRmap introduces a library of barcoded gRNAs into cells and couples in situ multiomic phenotyping (multiplexed immunofluorescence and RNA detection) with simultaneous cyclic in situ barcode detection and amplification. RCA-MERFISH first hybridizes probes to mRNAs or gRNA barcodes in fixed cells or tissues, ligates and performs RCA of those probes to generate localized DNA amplicons, then conducts sequential MERFISH readout cycles to image and decode multiplexed RNA identities in situ. Perturb-FISH combines MERFISH with localized amplification of gRNA sequences, allowing spatially resolved profiling of both perturbations and transcriptomes. PerturbView leverages IVT to amplify perturbation barcodes before ISS. Perturb-DBiT employs two methods for capturing gRNAs. The first is polyadenylation-based capture, where enzyme-mediated in situ polyadenylation adds a poly(A) tail to the 3′ end of the gRNA, enabling its detection. The second is direct capture, which uses a “capture” sequence complementary to the constant region of the gRNA scaffold to selectively bind and retrieve gRNAs. NIS-Seq produces bright sequencing signals directly from nuclear genomic DNA by leveraging T7 IVT to generate multiple RNA copies within the nucleus, followed by a cyclic ISS for readout. These spatial screening platforms support intracellular readouts in primary immune cells (optical phenotypes and transcriptional programs) and intercellular readouts (e.g., immune cell exclusion or infiltration within tissues), offering a powerful framework for dissecting spatially organized immune regulation and revealing biological insights in vivo. RT, reverse transcription; BC, barcode; CS, capture sequence; Trm, polymerase III terminator of a gRNA expression cassette; DBiT-seq, deterministic barcoding in tissue for spatial omics sequencing; NIS-Seq, nuclear in situ sequencing; RCA, rolling circle amplification; IVT, in vitro transcription; ISS, in situ sequencing.

Spatial CRISPR screens reveal intracellular and intercellular immune regulatory mechanisms. Building upon the Pro-Code method, Perturb-map was developed to integrate spatial information with CRISPR perturbations, uncovering how tumor clonality shapes the composition of the TME. CRISPRmap introduces a library of barcoded gRNAs into cells and couples in situ multiomic phenotyping (multiplexed immunofluorescence and RNA detection) with simultaneous cyclic in situ barcode detection and amplification. RCA-MERFISH first hybridizes probes to mRNAs or gRNA barcodes in fixed cells or tissues, ligates and performs RCA of those probes to generate localized DNA amplicons, then conducts sequential MERFISH readout cycles to image and decode multiplexed RNA identities in situ. Perturb-FISH combines MERFISH with localized amplification of gRNA sequences, allowing spatially resolved profiling of both perturbations and transcriptomes. PerturbView leverages IVT to amplify perturbation barcodes before ISS. Perturb-DBiT employs two methods for capturing gRNAs. The first is polyadenylation-based capture, where enzyme-mediated in situ polyadenylation adds a poly(A) tail to the 3′ end of the gRNA, enabling its detection. The second is direct capture, which uses a “capture” sequence complementary to the constant region of the gRNA scaffold to selectively bind and retrieve gRNAs. NIS-Seq produces bright sequencing signals directly from nuclear genomic DNA by leveraging T7 IVT to generate multiple RNA copies within the nucleus, followed by a cyclic ISS for readout. These spatial screening platforms support intracellular readouts in primary immune cells (optical phenotypes and transcriptional programs) and intercellular readouts (e.g., immune cell exclusion or infiltration within tissues), offering a powerful framework for dissecting spatially organized immune regulation and revealing biological insights in vivo. RT, reverse transcription; BC, barcode; CS, capture sequence; Trm, polymerase III terminator of a gRNA expression cassette; DBiT-seq, deterministic barcoding in tissue for spatial omics sequencing; NIS-Seq, nuclear in situ sequencing; RCA, rolling circle amplification; IVT, in vitro transcription; ISS, in situ sequencing.

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