Cited by 319CRISPR and Genetic EngineeringVirus-based gene therapy researchHematopoietic Stem Cell Transplantation

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CRISPR/Cas9 genome editing in human hematopoietic stem cells.

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Bak, Rasmus O, Dever, Daniel P, Porteus, Matthew H·2018·Nature Protocols

DOI 10.1038/nprot.2017.143PMC PMC5826598

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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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Authors: Bak, R. O., Dever, D. P., & Porteus, M. H.
Affiliations: Stanford Medicine (US)
Corresponding author: Porteus, Matthew H
ORCID: Bak, Rasmus O
Dever, Daniel P
Porteus, Matthew H
Venue: Nature Protocols
Publisher: Springer Science and Business Media LLC
Published: 2018-01-25
Volume: 13
Issue: 2
Pages: 358-376
ISSN: 1750-2799 (ISSN-L)
1754-2189
DOI: 10.1038/nprot.2017.143
PMC: PMC5826598
OpenAlex: W2784423079
Cited by: 319
References: 57
Citations by year: 2026: 23 · 2025: 37 · 2024: 31 · 2023: 47 · 2022: 35 · 2021: 51 · 2020: 50 · 2019: 31 · 2018: 13
FWCI: 12.88
Type: journal article
Language: en
Open access: green
www.ncbi.nlm.nih.gov
Licence: www.springer.com/tdm (tdm)
www.springer.com/tdm (vor)
Locations: Nature Protocols (publishedVersion)
PubMed (publishedVersion)
Europe PMC (PubMed Central) (acceptedVersion)
PubMed Central (acceptedVersion)
Funders: National Institutes of Health (5r01ai120766-02)
National Institute of Allergy and Infectious Diseases (R01-AI120766, R01AI097320)
Topics: Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
Health Sciences › Medicine › Hematology › Hematopoietic Stem Cell Transplantation
Keywords: CRISPR/Cas9 genome editing, hematopoietic stem cells, homologous recombination, AAV6 vector, Cas9 ribonucleoprotein, HSC engraftment, hematopoietic stem and progenitor cells, sickle cell disease, beta-thalassemia, primary immunodeficiency, gene therapy
MeSH terms: Gene Editing, Dependovirus, Animals, Flow Cytometry, Hematopoiesis, Hematopoietic Stem Cells, Humans, Genetic Therapy, Hematopoietic Stem Cell Transplantation, Gene Targeting, Polymorphism, Single Nucleotide, Mice, Homologous Recombination, CRISPR-Cas Systems

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CRISPR/Cas9 genome editing in human hematopoietic stem cells.

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