Abstract
Human CD34+ hematopoietic stem and progenitor cells (HSPCs) are a standard source of cells for clinical HSC transplantations as well as experimental xenotransplantation to generate “humanized mice”. To further extend the range of applications of these humanized mice, we developed a protocol to efficiently edit the genomes of human CD34+ HSPCs before transplantation. In the past, manipulating HSPCs has been complicated by the fact that they are inherently difficult to transduce with lentivectors, and rapidly lose their stemness and engraftment potential during in vitro culture. However, with optimized nucleofection of sgRNA:Cas9 ribonucleoprotein complexes, we are now able to edit a candidate gene in CD34+ HSPCs with almost 100% efficiency, and without affecting their potential for engraftment and multilineage differentiation in mice. The result is a humanized mouse from which we knocked out a gene of interest from their human immune system.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
This revision updates results with additional validation experiments both in vitro and in vivo, corrects several typos, and adds two authors.
