3019 – CRISPR-BASED ENGINEERING OF A REPORTER ALLELE DEMONSTRATES THAT HLF EXPRESSION DEFINES THE HUMAN HAEMATOPOIETIC STEM CELL STATE

Abstract

Haematopoietic stem cells (HSCs) sustain blood cell homeostasis throughout life and are able to regenerate all blood lineages following transplantation. Despite this clear functional definition, the accurate separation of human HSCs from their differentiated progeny can currently only be achieved through combinatorial measurement of multiple surface antigens. By extensive transcriptomic surveying of bulk and single-cell datasets, we have identified the master transcription factor HLF (Hepatic Leukemia Factor) as the most selective and consistently expressed marker gene in HSC-enriched sub-populations of different ontogenetic sources and ex vivo expanded CD34+ cord blood cells. To directly track its expression, we have engineered a genomic reporter strategy that relies on targeting its 3' non-coding region with a Cas9 ribonucleoprotein complex followed by infection with a recombinant AAV6 vector providing a repair template to introduce a fluorescent gene cassette. This approach routinely achieves high reporter insertion efficiency and is capable of selectively labeling the most immature human blood cells on the basis of a single engineered parameter. Most importantly, use of this reporter demonstrates that HLF-expressing cells comprise all of the stem cell activity in culture and in vivo during serial transplantation. Taken together, our results establish HLF as the most defining gene of the human haematopoietic stem cell state and outline a next-generation approach to mark these cells in real-time with unmatched fidelity.