CRISPR/Cas9 genome editing in human hematopoietic stem cells

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.

Efficient gene correction of an aberrant splice site in β‐thalassaemia iPSCs by CRISPR/Cas9 and single‐strand oligodeoxynucleotides

“…The great potential of iPSCs make us believe that utilizing them for gene correction can provide preclinical study for autologous cell therapy. Recent research demonstrated that combining CRISPR/Cas9 technology with rAAV6 can target human hematopoietic stem cells (HSCs) for gene repair via HDR, which offers an efficient technology for autologous cell therapy of β‐Thalassaemia . Moreover, iPSCs have the ability to differentiate into all cell types.…”

The rapidly advancing Class 2 CRISPR‐Cas technologies: A customizable toolbox for molecular manipulations

“…Applying CRISPR‐Cas9 under ex vivo conditions may be technically less challenging but equally valuable for disease therapy. For instance, ex vivo genome editing in hematopoietic stem cells (HSCs) could potentially provide a cure for patients carrying sickle cell disease (SCD) and β‐thalassaemia, and targeted insertions via CRISPR strategies may provide a valuable alternative to T‐cell engineering for cancer immunotherapy . A proof‐of‐concept study by Dewitt et al has reported an efficient correction of the E6V mutation at sickle alleles in patients' HSCs, through CRISPR‐HDR‐based replacement with the assistance of oligonucleotide donors.…”

Therapeutic gene editing in haematological disorders with CRISPR/Cas9

“…In CD34 + HSPCs, combinations of phenotypic markers, such as CD34 + , CD38 − , CD90 + , CD45RA − , CD49f + can identify a population where LT‐HSCs are enriched, but no definite set of markers uniquely pinpoints bona fide LT‐HSCs. This necessitates long‐term transplantation studies in immunodeficient mice to assess gene correction frequencies in the LT‐HSC population, which are the only cells that give rise to human haematopoietic repopulation several months after transplantation (Bak et al , ). Preferably, secondary transplants should be performed to assess true long‐term repopulation potential and editing frequencies.…”