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Updated: Sep 12, 2025
CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications
Published on: August 9, 2022
Protocol for efficient CRISPR/AAV-mediated genome editing and erythroid differentiation of human hematopoietic stem
Devesh Sharma1, Roshani Sinha1, Benjamin J Lesch1
1Department of Surgery, University of California, San Francisco, San Francisco, CA 94143, USA; Eli & Edythe Broad Center for Regeneration Medicine, University of California, San Francisco, San Francisco, CA 94143, USA.
Abstract:
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.
