Merck

Protocol for Efficient CRISPR/Cas9/AAV-Mediated Homologous Recombination in Mouse Hematopoietic Stem and Progenitor Cells.

STAR protocols (2020-07-21)
Ngoc Tung Tran, Janine Trombke, Klaus Rajewsky, Van Trung Chu
RESUMEN

Mutations that accumulate in self-renewing hematopoietic stem and progenitor cells (HSPCs) can cause severe blood disorders. To model such disorders in mice, we developed a CRISPR/Cas9/adeno-associated virus (AAV)-based system to knock in and repair genes by homologous recombination in mouse HSPCs. Here, we provide a step-by-step protocol to achieve high efficiency of gene knockin in mouse HSPCs, while maintaining engraftment capacity. This approach enables the functional study of hematopoietic disease mutations in vivo, without requiring germline mutagenesis. For complete details on the use and execution of this protocol, please refer to Tran et al. (2019).

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Sigma-Aldrich
DAPI, for nucleic acid staining
Sigma-Aldrich
Sodium phosphate monobasic, BioXtra, ≥99.0%
Sigma-Aldrich
Sodium phosphate dibasic, BioXtra, ≥99.0%
Sigma-Aldrich
Phenol red solution, 0.5%, liquid, sterile-filtered, BioReagent, suitable for cell culture