Protocol | Published:

CRISPR/Cas9 genome editing in human hematopoietic stem cells

Nature Protocols volume 13, pages 358376 (2018) | Download Citation

Abstract

Genome editing via homologous recombination (HR) (gene targeting) in human hematopoietic stem cells (HSCs) has the power to reveal gene–function relationships and potentially transform curative hematological gene and cell therapies. However, there are no comprehensive and reproducible protocols for targeting HSCs for HR. Herein, we provide a detailed protocol for the production, enrichment, and in vitro and in vivo analyses of HR-targeted HSCs by combining CRISPR/Cas9 technology with the use of rAAV6 and flow cytometry. Using this protocol, researchers can introduce single-nucleotide changes into the genome or longer gene cassettes with the precision of genome editing. Along with our troubleshooting and optimization guidelines, researchers can use this protocol to streamline HSC genome editing at any locus of interest. The in vitro HSC-targeting protocol and analyses can be completed in 3 weeks, and the long-term in vivo HSC engraftment analyses in immunodeficient mice can be achieved in 16 weeks. This protocol enables manipulation of genes for investigation of gene functions during hematopoiesis, as well as for the correction of genetic mutations in HSC transplantation–based therapies for diseases such as sickle cell disease, β-thalassemia, and primary immunodeficiencies.

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Acknowledgements

R.O.B. was supported through an individual postdoctoral grant (DFF-1333-00106B) and a Sapere Aude, Research Talent grant (DFF-1331-00735B), both from the Danish Council for Independent Research, Medical Sciences. D.P.D. was supported through a Stanford Child Health Research Institute (CHRI) grant and postdoctoral award. M.H.P. gratefully acknowledges the support of the Amon Carter Foundation, the Laurie Kraus Lacob Faculty Scholar Award in Pediatric Translational Research, and NIH grants R01-AI097320 and R01-AI120766. We further thank members of the Porteus and Majeti lab for helpful input, comments, and discussions.

Author information

Author notes

    • Rasmus O Bak
    •  & Daniel P Dever

    These authors contributed equally to this work.

Affiliations

  1. Department of Pediatrics, Stanford University, Stanford, California, USA.

    • Rasmus O Bak
    • , Daniel P Dever
    •  & Matthew H Porteus

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Contributions

R.O.B. and D.P.D. contributed equally to this work, as well as designed and performed most of the experiments necessary for the methodological development presented here. M.H.P. directed the research and participated in the design and interpretation of the experiments and the writing of the protocol. R.O.B. and D.P.D. wrote the manuscript with help from M.H.P.

Competing interests

M.H.P. is a consultant and has equity interest in CRISPR Tx, but CRISPR Tx had no input into the design, execution, interpretation, or publication of the methods or results herein.

Corresponding author

Correspondence to Matthew H Porteus.

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    Supplementary Figures 1–3 and the Supplementary Methods.

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DOI

https://doi.org/10.1038/nprot.2017.143

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