Abstract

In the human hematopoietic system, rare self-renewing multipotent long-term hematopoietic stem cells (LT-HSCs) are responsible for the lifelong production of mature blood cells and are the rational target for clinical regenerative therapies. However, the heterogeneity in the hematopoietic stem cell compartment and variable outcomes of CRISPR/Cas9 editing make functional interrogation of rare LT-HSCs challenging. Here, we report high efficiency LT-HSC editing at single-cell resolution using electroporation of modified synthetic gRNAs and Cas9 protein. Targeted short isoform expression of the GATA1 transcription factor elicit distinct differentiation and proliferation effects in single highly purified LT-HSC when analyzed with functional in vitro differentiation and long-term repopulation xenotransplantation assays. Our method represents a blueprint for systematic genetic analysis of complex tissue hierarchies at single-cell resolution.

Details

Title
Functional profiling of single CRISPR/Cas9-edited human long-term hematopoietic stem cells
Author
Wagenblast, Elvin 1   VIAFID ORCID Logo  ; Azkanaz, Maria 1 ; Smith, Sabrina A 1 ; Shakib, Lorien 1 ; McLeod, Jessica L 1 ; Krivdova, Gabriela 1 ; Araújo, Joana 1 ; Shultz, Leonard D 2 ; Gan, Olga I 1 ; Dick, John E 3   VIAFID ORCID Logo  ; Lechman, Eric R 1 

1  Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada 
2  The Jackson Laboratory, Bar Harbor, ME, USA 
3  Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada 
Pages
1-11
Publication year
2019
Publication date
Oct 2019
Publisher
Nature Publishing Group
e-ISSN
20411723
Source type
Scholarly Journal
Language of publication
English
DOI
https://doi.org/10.1038/s41467-019-12726-0
ProQuest document ID
2306794891
Copyright
© 2019. This work is published under http://creativecommons.org/licenses/by/4.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.