Primary human myeloid cells are emerging as important components of next-generation immunotherapies, but they have traditionally been difficult to engineer at the scale and precision needed for modern functional genomics. Monocytes, macrophages and dendritic cells are highly responsive to foreign nucleic acids and particles, and many standard gene-delivery methods can trigger toxicity, inflammation or loss of cellular function. A study published in Nature Biotechnology now describes a virus-like particle, or VLP, platform designed to overcome these limitations. The system delivers several forms of CRISPR machinery into primary human myeloid cells while maintaining cell viability and preserving their ability to respond to innate immune signals. The researchers also use the technology to perform pooled genetic screens and identify regulators of inflammatory behavior, including the central immune-modulating gene TNFAIP3.
VLPs are engineered particles that mimic key features of viruses without carrying a complete viral genome capable of producing infectious progeny. Their structure can be adapted to package and transport specific molecular cargo, allowing researchers to deliver genome-editing components directly into target cells. In the new work, the particles were used to deliver Cas9-based ribonucleoprotein complexes, in which the Cas9 nuclease is preassembled with a guide RNA. Once inside a cell, the guide RNA directs Cas9 to a matching genomic sequence, where the enzyme creates a targeted DNA break. Repair of that break can disrupt the gene, producing a knockout. Because the editing machinery is delivered as a transient protein-RNA complex rather than expressed continuously from a viral vector, the approach can limit the duration of Cas9 activity and reduce some of the complications associated with prolonged nuclease expression.
The researchers report that the VLP toolkit supports multiple CRISPR modalities in human monocytes, macrophages and dendritic cells. In addition to conventional gene knockout, the platform can deliver base-editing systems, which chemically convert individual DNA bases without generating the double-stranded breaks typically associated with standard Cas9 editing. Base editors can therefore create precise genetic substitutions, although their activity remains dependent on the location and sequence context of the target. The same delivery strategy was also used for epigenetic silencing. In this form of editing, CRISPR-guided regulatory proteins are directed to a gene’s control region and alter its transcriptional state without necessarily changing the underlying DNA sequence. Together, these capabilities give researchers a way to compare permanent gene disruption with more targeted nucleotide changes or reversible gene repression in the same broad class of immune cells.
The study further expands the system beyond editing existing genomic sequences. By combining VLP delivery with adeno-associated virus-mediated donor delivery, the researchers enabled site-specific insertion of large DNA sequences through homology-directed repair. In this configuration, the editing machinery creates a break at a chosen genomic site, while the AAV supplies a donor template containing the intended genetic payload and flanking regions that match the target locus. The cell can use these homologous sequences to copy the donor DNA into the genome. Such targeted integration is technically demanding in primary myeloid cells, which are generally resistant to manipulation and may respond strongly to DNA delivery. The combined platform could nonetheless support the introduction of larger genetic elements, a feature relevant to engineered-cell therapies that require the addition of receptors, regulatory circuits or other functional modules.
A central component of the work is SLICeVLP, a system developed to make pooled CRISPR screening possible in primary human macrophages. SLICeVLP separates two delivery tasks: guide RNA is supplied by a VPX-lentivirus, while Cas9 protein is delivered independently through engineered VLPs. This division allows the researchers to introduce guide sequences in a pooled format and then provide the nuclease as a controlled, transient editing reagent. In a pooled loss-of-function screen, thousands of cells can receive different guides targeting many genes, after which the population is exposed to a biological stimulus or sorted according to a measurable phenotype. Sequencing the guide identities in distinct cell populations reveals which genetic perturbations are associated with changes in that phenotype. The approach makes it possible to investigate gene function without having to create and maintain a separate engineered cell line for every candidate gene.
The researchers applied SLICeVLP to study the molecular controls governing tumor necrosis factor, or TNF, and CD80 expression in human macrophages. TNF is a potent inflammatory cytokine that helps coordinate immune responses but can also contribute to pathological inflammation when produced in excess. CD80 is a costimulatory molecule involved in communication between antigen-presenting cells and lymphocytes. By screening for genes that altered the levels of these markers, the team identified regulators of macrophage activation and inflammatory polarization. The work also incorporated Perturb-seq, a method that combines pooled genetic perturbations with single-cell RNA sequencing. Rather than measuring only one surface marker or secreted factor, Perturb-seq captures broad transcriptional changes in individual cells and links those expression profiles to the guide RNA that each cell received. This allows researchers to distinguish distinct cellular states and reconstruct how gene perturbations reshape immune programs.
The screen converged on TNFAIP3, which encodes the protein A20, as a central regulator of inflammatory polarization. A20 is widely recognized as a negative regulator of nuclear factor-κB-associated signaling, a pathway that controls the expression of many inflammatory genes. In the reported experiments, ablation of TNFAIP3 drove macrophages toward a strongly proinflammatory state. These cells were resistant to suppressive repolarization, suggesting that removing this regulatory brake did more than temporarily increase inflammatory gene expression. Instead, the perturbation appeared to stabilize a cellular program that could not readily be redirected by signals normally used to induce a less inflammatory phenotype. The result illustrates how pooled screening can expose control points that are difficult to identify through studies of individual genes or measurements limited to a single immune marker.
The researchers also examined the consequences of TNFAIP3 loss in chimeric antigen receptor macrophages, or CAR macrophages. These engineered cells are designed to recognize defined molecular targets through a synthetic receptor and then use macrophage effector functions to attack or process target material. In the study, removal of TNFAIP3 enhanced the cells’ cytotoxicity and reinforced their inflammatory behavior. The finding suggests that manipulating intrinsic signaling regulators could alter the activity of CAR macrophages after they encounter their targets. At the same time, the results highlight the balance required in therapeutic cell design: a stronger inflammatory and cytotoxic program may improve target-cell destruction, but persistent inflammatory activation could also affect safety, durability and interactions with surrounding tissues. The VLP platform provides a way to investigate these trade-offs systematically in primary human cells.
An important feature of the technology is that it was developed with the physiological properties of myeloid cells in mind. These cells are specialized to detect pathogens and foreign material, so delivery systems that work efficiently in more permissive cell types can provoke innate immune responses or impair survival in monocytes and macrophages. The study reports that VLP-mediated delivery maintained viability and preserved innate immune responsiveness, allowing the engineered cells to remain suitable for downstream functional testing. This distinction is essential for immunotherapy research because an editing method that changes a cell’s baseline activation state may produce misleading conclusions about gene function or therapeutic performance. By combining transient protein delivery, programmable guide RNAs and modular donor systems, the toolkit is intended to provide editing while retaining the biological behavior that makes primary myeloid cells valuable experimental and therapeutic models.
The platform does not eliminate the broader challenges of myeloid-cell engineering, including donor-to-donor variation, delivery efficiency across cell states and the need to assess unintended genomic or transcriptional effects. Nevertheless, the study establishes VLPs as a flexible delivery framework for knockout, base editing, epigenetic regulation and targeted DNA integration in primary human myeloid cells. Its use in pooled loss-of-function and Perturb-seq screens demonstrates how the same technology can move from molecular engineering to large-scale discovery. By connecting genetic perturbations with inflammatory phenotypes and therapeutic functions, the work offers a route to designing macrophage and other myeloid-cell therapies on the basis of systematic functional evidence rather than trial and error. The identification of TNFAIP3 as a key regulator of macrophage polarization further shows how these screens may reveal genetic interventions capable of reshaping the behavior of engineered immune cells.
Subject of Research: Virus-like particle-based CRISPR gene engineering and pooled functional-genomics screening in primary human myeloid cells.
Article Title: Virus-like particles enable targeted gene engineering and pooled CRISPR screening in primary human myeloid cells.
Article References: Jung, H., Devant, P., Ching, C. et al. “Virus-like particles enable targeted gene engineering and pooled CRISPR screening in primary human myeloid cells.” Nature Biotechnology (2026). https://doi.org/10.1038/s41587-026-03258-2
Image Credits: AI Generated
DOI: https://doi.org/10.1038/s41587-026-03258-2
Keywords: Virus-like particles, CRISPR, primary human myeloid cells, monocytes, macrophages, dendritic cells, VLP-mediated delivery, base editing, epigenetic silencing, homology-directed repair, AAV, SLICeVLP, Perturb-seq, TNFAIP3, CAR macrophages, immunotherapy.
Tags: CRISPR-Cas9 delivery systemsfunctional genomics in immune cellsgene delivery methodsgenome editing in monocytesimmune response preservationImmunotherapyinflammation regulationmacrophages and dendritic cellspooled CRISPR screensprimary human myeloid cellstargeted gene engineeringVirus-like particles
