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

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

Summary: 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 an...

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Bibliographic Details
Main Authors: Devesh Sharma, Roshani Sinha, Benjamin J. Lesch, M. Kyle Cromer
Format: Article
Language:English
Published: Elsevier 2025-09-01
Series:STAR Protocols
Subjects:
Cell Biology
Cell culture
Flow Cytometry
Molecular Biology
CRISPR
Stem Cells
Online Access:http://www.sciencedirect.com/science/article/pii/S2666166725004241
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Summary:Summary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
ISSN:2666-1667

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