CRISPR/Cas9 genome editing in human hematopoietic stem cells.

Bak RO, Dever DP, Porteus MH
Source: Nat Protocols
Publication Date: (2018)
Issue: 13(2): 358-376
Research Area:
Immunotherapy / Hematology
Cells used in publication:
CD34+ cell, human
Species: human
Tissue Origin: blood
Nucleofector™ I/II/2b
4D-Nucleofector™ X-Unit

CD34+ were thawed or isolated and cultured for 2d before Nucleofection. 0.5-1.0 x10e6 cells were transfected with Nufe 2b program U-014 (t cell nucleofector solution) or with the 4D Nuclefoector and protgram DZ-100 and P3 solution. Please find more details in the protocol itself. 


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, ß-thalassemia, and primary immunodeficiencies.