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J Exp Med (4): Spatial CRISPR Screening—Unveiling the Regulatory Codes of the Tumor Microenvironment in Situ within Tissues

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This article is “The Next Generation” CRISPR Chapter Four of “Screening and Causal Systems Immunology.”

J Exp Med : Why do we need “ Causal System Immunology ”？ ——CRISPR The historical context of screening

J Exp Med (2): Bulk CRISPR Screening platform —— From T Functional dissection from the cellular level to the entire immune system

J Exp Med (3): Single cell CRISPR Filter —— Decoding Immune Heterogeneity and Causal Networks at Single-Cell Resolution

The first three articles respectively introduce the overall framework, Bulk CRISPR Screening and single cells CRISPR Through screening, we have gradually come to understand how genes influence cell fate. Yet a crucial dimension has consistently been missing. —— Location—under the same cellular and genetic perturbations, different microenvironments may lead to entirely distinct outcomes. Spatiality. CRISPR Filter ( Spatial CRISPR Screening The emergence of ) is precisely aimed at answering this question: how exactly does a cell’s spatial location determine its behavior and fate?

01  When the organization is disrupted, causal information is lost as well.

Looking back at the previous two generations CRISPR A common prerequisite for screening techniques is that, prior to analysis, tissues must first be dissociated into single cells. This is indispensable for single-cell sequencing; however, it also causes researchers to lose a critically important piece of information. —— Spatial relationship In fact, a real tumor is never a random mixture of various cell types: CD8⁺ T Cells aggregate in specific regions; cancer-associated fibroblasts ( CAF ) Forming a physical barrier; tumor-associated macrophages exhibit spatial distribution preferences; the vascular network, hypoxic regions, and immunosuppressive niches collectively constitute a complex tissue architecture. These spatial structures together comprise the tumor microenvironment ( TME ).

Tumor progression, immune evasion, and therapeutic response often occur within these spatial architectures. Once tissues are dissociated, researchers can still determine which genes a given cell expresses, but they lose the ability to reconstruct its original location or to infer the cellular interactions it previously engaged in. As a result, an essential dimension is lost. 02 Paradigm Shift: From What Happened to Why It Happened

 

From Looking at the evolution of CRISPR screening, the questions being asked are steadily becoming more complex. Bulk CRISPR focuses on: which gene is important? Single-cell CRISPR goes a step further, asking: how does this gene alter the cellular state? Meanwhile, spatial CRISPR begins to pose a new question: why does this change occur precisely here? What may seem like merely the addition of a spatial dimension actually signals a shift in the research paradigm. This is because an increasing number of tumor‑immune phenomena fall under the category of classic non‑cell‑autonomous regulation.

 

When a cell undergoes genetic alterations, the effects often extend beyond itself. It may alter the state of neighboring macrophages by secreting cytokines; it may also impede… by remodeling the extracellular matrix. T-cell infiltration; it may also alter the entire local immune microenvironment by modulating the signaling networks of neighboring cells. In other words, what is truly altered may not be a single cell, but rather the entire microenvironment. Meanwhile, conventional CRISPR… Screening, however, struggles to capture such cross-cellular and cross-spatial causal relationships.

03  Space CRISPR : Reintroduce genetic perturbations into their native tissue context

 

Space The core idea of CRISPR screening is remarkably straightforward: rather than isolating cells from a tissue for study, it involves observing the outcomes of perturbations directly within the native tissue context. On a single tissue section, one can simultaneously obtain three critical types of information: which gRNA a cell carries, what molecular state the cell is in, and where the cell is located within the tissue. When these three layers of information are integrated, the object of study ceases to be an individual cell and instead becomes a comprehensive system defined by perturbation × state × spatial environment.

 

For the first time, researchers have been able to directly observe whether, upon gene knockout, the surrounding immune cells undergo changes; whether local immune architectures are remodeled; and whether communication between the tumor and its microenvironment is reprogrammed. Spatial Thus, CRISPR has become an important bridge linking functional genomics and space biology.

04  Space CRISPR How to precisely visualize genetic perturbations

 

How can we accurately identify the genetic perturbations carried by each cell while preserving spatial coordinates? To address this core technical bottleneck, scientists have undertaken several rounds of efficient paradigm shifts in engineering:

 

First-generation exploration : Let disturbance and space meet for the first time.

Representative platform :Perturb-map

Utilize Pro-Code Protein barcodes are labeled differently. gRNA and, in conjunction with spatial transcriptomics, analyzed tumor tissues. For the first time at the genomic level… Weave at the regional level (~55 µm) It has achieved a spatial mapping of which gene is perturbed and the microenvironment that forms around it, and established a perturbation framework. - Clone - A framework for the relationships among spatial locations.

 

Second-generation upgrade : Entering the era of subcellular resolution

Representative platform :Perturb-FISH/CRISPRmap/RCA-MERFISH

As MERFISH Spatial imaging and rolling circle amplification (RCA) The technology is mature, and the technology platform has begun to enable high-definition data retrieval directly within organizations. gRNA Identity. With spatial resolution advancing to the subcellular level, researchers can not only observe phenotypic changes but also precisely pinpoint their locations, thereby gaining the true capability to dissect intercellular communication networks in situ.

 

Third-generation crossover : Moving toward whole-genome throughput and clinical compatibility

Representative platform :Perturb-DBiT/PerturbView/NIS-Seq

This generation of platforms, employing strategies such as in situ amplification, direct capture, and intranuclear signal amplification, has achieved significant improvements in both throughput and detection range. rise. Among them, with Perturb-DBiT Breakthrough technologies, exemplified by ..., have not only achieved genome-wide-scale… CRISPR Screening and whole-transcriptome analysis offer more comprehensive compatibility with the most challenging clinical cases. FFPE( Paraffin embedding ) Sample, marking space CRISPR It has formally expanded from mechanistic research to a large-scale, clinical‑sample‑based application framework.

 

| Overview of Mainstream CRISPR Platforms in the Space Sector

05  Space CRISPR What exactly has been broken through?

Space CRISPR The most important value lies not merely in obtaining higher-resolution data, but in beginning to address biological questions that were previously unanswerable.

 

① Revealing the spatial organization principles of the tumor microenvironment

Space CRISPR For the first time, it has explained the tumor microenvironment at the causal level ( TME ) Sources of heterogeneity. Studies have shown that distinct tumor clones can establish heterogeneous local immune microenvironments by secreting different extracellular factors, thereby giving rise to specific immune niches ( Immune Neighborhood ). These spatial structures further determine T Cellular infiltration, immune activation, and response to immunotherapy. With IL-4 Ligands represented by - Receptor–ligand interactions can even reshape the local immune landscape, leading to the development of therapeutic resistance.

 

② Discovery of novel immunotherapy targets

Unlike traditional screening, which primarily focuses on the intrinsic effects of cells, spatial… CRISPR A new class of regulatory factors has begun to be discovered. —— These are genes that exert their effects through paracrine signaling, intercellular communication, and spatial organization. Although they may not directly alter the tumor cells themselves, they can profoundly shape the entire immune microenvironment, thereby emerging as promising new therapeutic targets.

 

③ From the Tumor Microenvironment to Inter‑Tissue Communication

Space CRISPR Its applications are not limited to cancer. An increasing number of studies are focusing on immune communication across different tissues. For example, research has shown that signals originating from the liver can promote the engraftment of disseminated tumor cells, suggesting that the metastatic process is jointly regulated by cross‑tissue microenvironments. In the future, spatial… CRISPR It holds promise for further elucidating causal regulatory networks at the tissue scale. 06  How far are we from a complete spatiotemporal causal network?

 

Despite rapid development, space CRISPR It remains in the early stages. At present, the greatest challenges stem from four main areas. First, the complex cellular heterogeneity within tissues continues to hinder the precise mapping of perturbations to phenotypes. Second, most experiments still yield only static snapshots, making it difficult to capture the dynamic changes that underpin immune regulatory processes. Third, a trade-off persists between high resolution and high throughput: higher resolution typically limits the scale of genes that can be interrogated, while broader coverage often comes at the expense of spatial precision. Finally, the integrated analysis of multimodal spatial data remains a major challenge in computational biology. Establishing robust causal models of intercellular relationships is still a long way off. 07  Conclusion

 

If we say Bulk CRISPR Let’s identify the key genes, single-cell. CRISPR Let us understand cell fate, then space. CRISPR Then let us begin to understand: Why do these fates unfold in specific contexts? At this point, CRISPR The screening has achieved a significant leap forward. The objects of study are no longer limited to genes or cells alone; they now encompass the intricate ecosystem composed of genes, cells, and the microenvironment. For tumor immunology, this signifies a shift from investigating intracellular regulatory mechanisms to examining causal networks at the tissue scale—bringing us one step closer to a truly systems‑level understanding of immunity. 08 Next Episode Preview

 

Spatial information fills in the organizational dimension, but the immune system’s functioning has never been determined by a single gene. In the next installment, we will turn to the final topic of this series: Combination CRISPR Perturbation Screening (Combinatorial CRISPR Screening) — When multiple genes are perturbed simultaneously, how can we unravel the causal relationships embedded within gene networks?

 

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