Protocol for efficient CRISPR/AAV-mediated genome editing and erythroid differentiation of human hematopoietic stem and progenitor cells.

Here, we present a protocol for genome editing in human hematopoietic stem and progenitor cells (HSPCs) using CRISPR-Cas9 ribonucleoproteins and adeno-associated virus (AAV)-mediated homology-directed repair. We describe steps for AAV production, purification, and titration; HSPC thawing and culture; genome editing; and quantification of editing frequencies. We then detail procedures for erythroid differentiation assays. This protocol ensures high editing efficiency while maintaining cell viability and engraftment potential. For complete details on the use and execution of this protocol, please refer to Chu et al.1.