Reversion to quiescence preserves hematopoietic stem cells following genome editing. Mus musculus

Other than genetically engineered mice, there is a lack of reliable platforms to study quiescence of hematopoietic stem cells (HSCs). Here, we established a non-viral genome editing method optimized for mouse HSCs based on the CRISPR-Cas9 system and used it in combination with a culture condition that maintains quiescence. We demonstrated that edited HSCs can be reverted to quiescence using only physiological factors found in bone marrow. Edited HSCs in the quiescence-maintaining condition retained their phenotypes and functions better than those in the proliferative condition. By applying this approach, we confirmed that loss of glucose-6-phosphate isomerase 1 (Gpi1) or glyceraldehyde-3-phosphate dehydrogenase (Gapdh), which are glycolytic enzymes, severely impaired proliferative HSCs, but this effect was minimized in quiescent HSCs due to mitochondrial compensation. Our results show that an HSC-optimized genome editing method in combination with quiescence-maintaining culture will help to analyze quiescence of HSCs ex vivo.