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

STAR Protocols
Ngoc Tung TranVan 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).

References

Aug 4, 2010·Methods in Molecular Biology·Dmitry Y GuschinEdward J Rebar
Aug 30, 2012·Nature Methods·Caroline A SchneiderKevin W Eliceiri
Jan 17, 2016·BMC Biotechnology·Van Trung ChuRalf Kühn
Nov 3, 2016·Proceedings of the National Academy of Sciences of the United States of America·Van Trung ChuKlaus Rajewsky
Dec 12, 2018·Molecular Therapy. Methods & Clinical Development·Marti Cabanes-CreusLeszek Lisowski

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Methods Mentioned

BETA
PCR
transfection
FACS
flow cytometry
flow
gene knockin

Software Mentioned

FlowJo
FACS analyzer
ImageJ
Synthego
CrispRGold

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