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J Exp Med (5): Combined CRISPR Perturbation Screening—When Multiple Genes Are Altered Simultaneously, How Do We Unravel the True Causal Networks of Living Systems?

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This article is “The Next Generation” CRISPR The fifth installment of the “Screening and Causal Systems in Immunology” series, and also its concluding chapter.

As CRISPR With advances in screening technologies, researchers can now identify key gene functions, characterize cellular states, and visualize tissue-level changes following perturbations. Single-gene–level atlases are steadily being refined. However, as investigations deepen, a more complex reality is emerging: many biological processes cannot be fully accounted for by a single gene or a single signaling pathway. Against this backdrop, the focus of research is shifting from isolated gene functions to the coordinated interplay of multiple genes and their combinatorial effects. CRISPR Disturbance screening ( Combinatorial CRISPR Screening ), precisely emerged during this transformation, seeking to address a higher-order question: How does a system reconfigure its operational mode when multiple genes are perturbed simultaneously?

01 When single genes can no longer account for biological systems, research begins to encounter new bottlenecks.

Over the past decade, CRISPR Screening has helped researchers identify a large number of genes with critical functions. However, an increasing number of experiments are revealing a common phenomenon: when many genes are knocked out, virtually no phenotypic changes are observed in the cells. PD-1 Missing, T Cellular function recovery is limited; even after a drug‑resistance gene is deleted, the tumor can still continue to grow; and when one signaling pathway is inhibited, a new bypass quickly re‑establishes itself. These findings do not imply that these genes are without function; rather, they demonstrate that living systems possess an extraordinary capacity for… Network redundancy and resilience.

When one gene is inactivated, another immediately compensates; when a pathway is blocked, new regulatory networks are rapidly reconfigured. What truly determines disease progression is no longer a single gene, but the entire genetic interaction network. Genetic Interaction Network ). Thus, a new question arises: if living systems are themselves networks, why do we still rely on single-gene experiments to understand them?

02 The first three generations CRISPR The screening process continues to evolve, but the underlying premise remains unchanged.

CRISPR Screening technologies have advanced rapidly. From bulk‑level screening to single‑cell resolution and then to spatial, in situ detection, researchers are gaining increasingly rich information and achieving ever higher resolution in their observations of living systems. Nevertheless, regardless of how these technologies evolve, their core research paradigm remains unchanged: perturb a single gene and observe the resulting biological changes.

This strategy has greatly advanced the field of functional genomics and enabled researchers to establish numerous causal links between genes and phenotypes. However, as research has increasingly shifted toward the tumor microenvironment, immune regulation, and complex diseases, it has become clear that relying solely on single-gene perturbations is increasingly inadequate for explaining the intricate phenomena observed in living systems. The emergence of drug resistance, the establishment of immune evasion, and the synergistic effects of combination therapies are rarely the result of a single gene acting in isolation; rather, they arise from the concerted involvement of multiple regulatory pathways, which compensate for one another and engage in dynamic feedback loops.

In other words, the first three generations CRISPR Screening has continually broadened the depth of our observations of living systems, yet it has always been grounded in… The Causal View of the Monogenetic Center However, what is truly lacking is a systematic dissection of the interactions among genes.

03 Combination CRISPR : Unlocking Gene Regulatory Networks

Combination CRISPR The core idea is very simple: If living systems depend on networks to function, then experiments should target those networks directly. Around this goal, the combination CRISPR It has undergone several significant evolutions.

The earliest to appear was Gene screening query ( Query-based Screening ). Researchers first fix a query gene and then perform genome-wide screening to identify a second gene that exhibits either synergistic or antagonistic interactions with it. This strategy was the first to enable genetic interaction screening; however, each time a new query gene is introduced, an entire screening round must be repeated, limiting its scalability.

Subsequently appeared Dual gene CRISPR Filter ( dKO ). Researchers utilized a dual… gRNA In the library, two genes are simultaneously knocked out within the same cell, and the system identifies synergistic, antagonistic, and synthetic lethal interactions ( Synthetic Lethality ) and other genetic interaction relationships. For example, when two genes are individually knocked out, they have little to no effect; however, their simultaneous inactivation leads to cell death—this has become a key theoretical foundation for combination anti‑tumor therapies in recent years.

However, as the number of genes requiring study continues to grow, new challenges are once again emerging. If research… 100 A single gene, when combined in pairs, approaches 5000 Species; if research 1000 With a single gene, the number of possible combinations can reach hundreds of thousands or even millions. The number of combinations has begun to grow exponentially. , Tradition Cas9 The system can no longer cope.

04 Technology has entered the true polygenic era.

Combination The advancement of CRISPR has propelled experimental tools into a true multi‑gene era. Cas12a enables simultaneous perturbation of multiple genes within the same cell via gRNA arrays, while CLASH and Cas12a‑mediated transgenic mice further extend this capability to high‑throughput in vitro screening and in vivo editing of immune cells, allowing complex genetic networks to be directly manipulated in physiological contexts. Meanwhile, Cas13d extends perturbation from the DNA to the RNA level, CaRPool‑seq integrates single‑cell sequencing to dissect cellular state changes under multiplexed perturbations, and MAGIC employs AAV‑mediated delivery of multi‑target RNAs to achieve concurrent regulation of multiple immune‑suppressive molecules. With these advances, combinatorial CRISPR has achieved a technological leap—from two‑gene screening to the perturbation of entire multi‑gene networks.

This capability enhancement leads to an exponential expansion of the design space, shifting the focus from how to conduct perturbation experiments to how to interpret vast volumes of perturbation data, thereby elevating computational analysis to a core infrastructure. Luo Wait ( 2025 ) Proposes dual‑action immunotherapy targets and reveals that the same gene in tumor cells and… T Opposing functions within the cell, thereby achieving simultaneous inhibition of immune evasion and enhancement. T A one‑two punch strategy for cell killing.

Meanwhile, pertpy 、 crispat and CLEANSER Gradually build up scCRISPR Computational framework, used respectively for perturbation integration analysis, gRNA Data correction and environmental noise removal jointly enable the reliable analysis of large-scale composite perturbation datasets. Ultimately, the composite… CRISPR The core shift: the focus of research has moved from the function of individual genes to the causal networks formed by gene–gene interactions themselves.

| Technical Advantage Comparison of Combined Perturbations

05 Future Prospects: A Comprehensive Map of Causal Systems Immunology

Shi & Chi The envisioned future depicts a combination… CRISPR The ultimate goal is not merely to scale up the perturbation, but to construct a predictive causal graph of the immune system. Causal Perturbation Atlas of Immunity ), enabling the system’s response to genetic perturbations to be modeled and interpreted in a holistic manner.

With advances in technology, whole-genome perturbation maps will systematically characterize the perturbation‑response dynamics of immune cells under both homeostatic and disease states. This endeavor is analogous to the Human Genome Project, but shifts the focus from sequence information to the causal response space.

In vivo genetic interaction screening is further advancing multi‑gene combination therapies, shifting them from empirical combinatorial approaches to structured design and gradually transforming combination treatment from being experience‑driven to network‑driven. At the same time, the scope of research has expanded beyond tumor systems to encompass cross‑tissue communication networks, with the central challenge lying in systematically dissecting inter‑tissue signaling regulators, microenvironmental markers, and in vivo… CRISPRa The strategy provides a new technological pathway for this.

These directions ultimately converge into a single paradigm shift: immunological research is moving away from observation… — Suppose — Single-gene validation, moving toward systematic perturbation — Causal Network Reconstruction — Computational Integration — Target discovery — Precision treatment design. At its core, this represents a fundamental shift: the immune system is being reimagined from a collection of gene functions into a causal network that can be perturbed, modeled, and predicted.

06 Conclusion

Looking back at the entire series, CRISPR The evolution of screening is not merely a matter of technological iteration; it represents the ongoing refinement of immunological research paradigms. As genetic perturbations become increasingly integrated with single-cell multi-omics, spatial omics, combinatorial screening, and computational analysis, CRISPR Screening is evolving from an experimental tool for elucidating gene functions into a core methodology for reconstructing the causal regulatory networks of complex living systems.

As stated in the… Next-generation CRISPR screens enable causal systems immunology As envisioned, the future of immunology research will aim not merely to identify novel functional genes, but to elucidate the causal mechanisms underlying the entire immune system and to leverage this understanding to guide more precise and efficacious immunotherapies. From functional screening to causal dissection, from single-gene studies to the reconstruction of systemic networks, CRISPR Screening is driving immunology toward causal systems immunology ( Causal Systems Immunology ) of the new era.

07 Series Article Index

Part One - Introduction and Background

The concept of causal system immunology, CRISPR Toolbox Overview and Four Major Application Areas

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

> Part Two -Bulk CRISPR screening

New editing tools (base editing, prime editing, RNA Editor), in vivo screening strategies, applications across multiple cell types

J Exp Med (2): Bulk CRISPR Screening platform —— From T Functional Analysis from the Cellular Level to the Entire Immune System

> Part Three - Single-cell CRISPR screening

gRNA Evolution of capture technologies, multimodal readout platforms, T Cell Fate Causal Network

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

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

Perturb-map to Perturb-FISH Wait for the new platform, TME Spatial Regulation Mechanism