Sequential in vivo CRISPR screens identify the clonal dominance of Nf1 loss in long-term hematopoiesis

Systematically interrogating the genetic regulators of long-term hematopoietic stem cell (HSC) function and their roles in malignant transformation in vivo remains a central challenge. We developed and validated a sequential in vivo CRISPR screening platform characterized by high sgRNA recovery after a 5-month reconstitution period and a pioneering pooled secondary transplantation strategy. This system not only identified enriched genes but also modeled the dynamics of clonal evolution. As a robust validation of our platform’s sensitivity, the screen identified Neurofibromin 1 (Nf1 ), a quintessential tumor suppressor, as the dominant hit using a focused transcription factor library. The primary screen recapitulated a myeloproliferative state. Strikingly, the secondary transplantation screen amplified this phenotype and led to malignant hematopoiesis. Thus, this 2-step approach establishes a powerful paradigm for studying the genetic drivers of long-term hematopoiesis and the evolutionary dynamics of hematological malignancies.