Single-cell CRISPR screen maps epigenetic regulators of lineage specification and malignancies in human HSPCs

Mapping how genetic perturbations influence cell fate is key to understanding human development and disease. Here, we developed a massively parallel single-cell CRISPR (scCRISPR) screening platform in primary human hematopoietic stem and progenitor cells (HSPCs), profiling 322 regulators across 172,382 single-perturbed cells. The resulting multi-layered regulatory framework captured known associations and enabled functional annotation of regulator-phenotype relationships. Specifically, our atlas revealed that regulators acted within perturbation clusters to govern distinct gene programs that shaped cell fate. As proof of concept, we illustrated several examples. SMYD3 emerged as a negative regulator of long-term regenerative capacity. Analysis of lineage output further identified KMT5B, TRIM24, SETD7, and NSD3 as regulators associated with lineage-biased outputs. Finally, integration with clinical datasets identified PRDM10 as a candidate dependency in acute myeloid leukemia (AML). This work establishes a scalable functional genomics platform in a primary human stem cell system.