Genome‑wide replication‑competent HIV‑guided CRISPR screen identifies DNA metabolism/repair regulators as antiviral factors

Innate antiviral factors are critical components of host defense. However, many physiologically relevant mechanisms restricting HIV-1 replication remain to be identified. To uncover such factors, we developed an HIV-1–guided CRISPR-Cas9 screen using replication-competent HIV-1 libraries encoding 77,441 distinct sgRNAs targeting all annotated human genes. Viral propagation in the presence or absence of IFN-β enabled the identification of multiple cellular genes that significantly restrict HIV-1 replication. Among the top hits were factors involved in DNA metabolism and repair (DNase1L2, NTHL1, POLE4, RMI2), chromatin regulation (MPND), and innate immune signaling (GNB1L, TRIM9). Their restrictive effects were validated in primary CD4⁺ T cells, confirming relevance in HIV-1’s major target cells in vivo. Together, these findings establish genome-wide replication-competent HIV-1 CRISPR screening as a powerful approach to identify restriction factors and uncover a previously unrecognized role of genome integrity pathways in cell-intrinsic antiviral immunity.