CRISPR-based functional genomics for dissecting therapeutic dependency in primary acute myeloid leukemia samples

Cancer functional genomics enables high-throughput target discovery and mechanistic investigation, yet its application has remained largely confined to mouse models and established human cancer cell lines. Direct functional interrogation of heterogeneous primary tumors offers a powerful opportunity to evaluate therapeutic targets and uncover cancer dependencies or resistance mechanisms. Here, we developed an optimized CRISPR-based platform for functional genomics in patient-derived xenograft and primary acute myeloid leukemia (AML) samples harboring diverse pathogenic mutations. Integrated in vitro and in vivo CRISPR-Cas9 knockout and CRISPR interference (CRISPRi) dropout screens validated known AML-biased targets and identified cis- regulatory elements essential for leukemic growth. Coupling pooled CRISPR perturbations with single-cell RNA sequencing (Perturb-seq) further resolved the perturbation-induced alterations in regulatory networks, cell cycle states, and cellular hierarchies in primary AML samples. Together, these studies establish a general and robust framework for leveraging CRISPR-based functional genomics to directly dissect cancer dependencies and cellular heterogeneity in primary AML patient samples. • Optimized CRISPR platform for genetic studies in primary AML samples • In vitro and in vivo CRISPR screens reveal shared and distinct AML dependencies • CRISPRi screens identify essential cis- regulatory elements in PDXs • Perturb-seq dissects regulatory networks and heterogeneity in primary AML cells Direct CRISPR functional genomics in primary patient samples holds promise for therapeutic target validation and discovery. Cao et al. develop an optimized CRISPR platform for genetic screening in primary acute myeloid leukemia samples. Integrated knockout, knockdown, and Perturb-seq screens dissect cancer vulnerabilities and cellular heterogeneity directly in patient-derived cells.