Precision gene editing in primary hematopoietic stem and progenitor cells (HSPCs) would facilitate both curative treatments for monogenic disorders as well as disease modelling. Precise efficiencies even with the CRISPR/Cas system, however, remain limited. Through an optimization of guide RNA delivery, donor design, and additives, we have now obtained mean precise editing efficiencies >90% on primary cord blood HSCPs with minimal toxicity. Critically, editing is even across the progenitor hierarchy, and does not substantially distort the hierarchy or affect lineage outputs in colony-forming cell assays. As modelling of many diseases requires heterozygosity, we also demonstrated that the overall editing and zygosity can be tuned by adding in defined mixtures of mutant and wild-type donor. With these optimizations, editing at near-perfect efficiency can now be accomplished directly in human HSPCs. This will open new avenues in both therapeutic strategies and disease modelling.

No abstract