Virus-like particles enable targeted gene engineering and pooled CRISPR screening in primary human myeloid cells

Primary human myeloid cells are promising candidates for immunotherapy, yet efficient and scalable technologies for genetic engineering and screening in these cells are limited. Here we present a virus-like particle (VLP)-based toolkit that delivers diverse CRISPR genome editing modalities to human monocytes, macrophages, and dendritic cells with high efficiency while preserving viability and innate immune responsiveness. VLP-mediated delivery of ribonucleoprotein payloads supports gene knockout, base editing, and epigenetic silencing. Furthermore, in combination with AAV-mediated donor delivery, this approach enables site-specific integration of large DNA sequences via homology-directed repair. We also developed SLICeVLP, a system combining sgRNA delivery via VPX-lentivirus with Cas9 protein delivery via engineered virus-like particles (eVLPs), and applied it to perform pooled loss-of-function screens and Perturb-seq in human macrophages. We uncovered regulators of TNF production and CD80 expression in human macrophages, converging on TNFAIP3 as a central regulator of inflammatory polarization. TNFAIP3 ablation promoted a pro-inflammatory cell state that is resistant to suppressive polarization, and augmented cytotoxicity in engineered HER2 CAR-macrophages. Taken together, this platform enables unbiased functional genomics in primary human myeloid cells, with direct implications for myeloid cell therapy design.