A Single Cell Cloning Platform for Gene Edited Functional Murine Hematopoietic Stem Cells

Gene editing using engineered nucleases frequently produces on- and off-target indels in hematopoietic stem cells (HSCs). Gene-edited HSC cultures thus contain genetically heterogenous populations, the majority of which either do not carry the desired edit or harbor unwanted mutations. In consequence, transplanting edited HSCs carries the risks of suboptimal efficiency and of unwanted mutations in the graft. Here, we present an approach for expanding gene-edited HSCs at clonal density, allowing for genetic profiling of individual clones before transplantation. We achieved this by developing a defined, polymer-based expansion system and identifying long-term expanding clones within the CD201 + CD150 + CD48 - c-Kit + Sca-1 + Lin - population of pre-cultured HSCs. Using the Prkdc scid immunodeficiency model, we demonstrate that we can expand and profile edited HSC clones to check for desired and unintended modifications. Transplantation of Prkdc- corrected HSCs rescued the immunodeficient phenotype. Our ex vivo -manipulation platform establishes a novel paradigm to control genetic heterogeneity in HSC gene editing and therapy.