Protocol for optimizing culture conditions for ex vivo activation during CRISPR-Cas9 gene editing in human hematopoietic stem and progenitor cells
Long-range correction strategies require ex vivo activation of hematopoietic stem and progenitor cells (HSPCs) to engage the homology-directed repair (HDR) mechanism, but prolonged culture causes harmful cellular responses, reducing the long-term functionality of gene-edited (GE) HSPCs. Here, we present a protocol for optimizing culture conditions for ex vivo activation during CRISPR-Cas9 gene editing in human HSPCs. We describe steps for HSPC thawing, ex vivo treatments, gene editing, and downstream in vitro and in vivo analyses to assess the functionality of GE-HSPCs. For complete details on the use and execution of this protocol, please refer to della Volpe et al.<sup>1</sup>.
Authors
- Raffaella Di Micco (ORCID: https://orcid.org/0000-0002-0639-8238)
- Lucrezia della Volpe (ORCID: https://orcid.org/0000-0002-9087-1293)
- Roberta Vacca
Institutions
- The San Raffaele Telethon Institute for Gene Therapy (IT)
Publication Details
- Journal
- STAR Protocols
- Published
- 2025-04-02
- DOI
- https://doi.org/10.1016/j.xpro.2025.103722
- Primary Topic
- CRISPR and Genetic Engineering
- Type
- article
- Field-Weighted Citation Impact
- 0.00
Funders
- New York Stem Cell Foundation
- Human Frontier Science Program
- Human Frontier Science Program
- Associazione Italiana per la Ricerca sul Cancro
- European Research Council
- H2020 European Research Council
- HORIZON EUROPE European Innovation Council