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

doi:10.1016/j.xpro.2020.100028

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

STAR Protoc. 2020 Jun 19;1(1):100028. doi: 10.1016/j.xpro.2020.100028.

Authors

Ngoc Tung Tran¹, Janine Trombke¹, Klaus Rajewsky¹, Van Trung Chu¹

Affiliation

¹ Immune Regulation and Cancer, Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association, Berlin 13125, Germany.

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).

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
CRISPR-Cas Systems*
Gene Knock-In Techniques / methods*
Hematopoietic Stem Cells / cytology*
Homologous Recombination*
Mice
Stem Cells / cytology*