Protocol for Efficient CRISPR/Cas9/AAV-Mediated Homologous Recombination in Mouse Hematopoietic Stem and Progenitor Cells
Ngoc Tung Tran, Janine Trombke, Klaus Rajewsky, Van Trung Chu
Abstract
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).
Topics & Concepts
CRISPRHomologous recombinationHaematopoiesisGermlineBiologyProgenitor cellMutagenesisCas9Homologous chromosomeGenome editingGeneStem cellGeneticsMutationCRISPR and Genetic EngineeringPluripotent Stem Cells ResearchSingle-cell and spatial transcriptomics