Near-perfect precise on-target editing of human hematopoietic stem and progenitor cells

Author:

Cloarec-Ung Fanny-Mei1ORCID,Beaulieu Jamie1,Suthananthan Arunan1,Lehnertz Bernhard1,Sauvageau Guy1,Sheppard Hilary M12,Knapp David JHF13ORCID

Affiliation:

1. Institut de Recherche en Immunologie et en Cancérologie, Université de Montréal

2. School of Biological Sciences, Faculty of Science, University of Auckland

3. Département de Pathologie et Biologie Cellulaire, Université de Montréal

Abstract

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 and without observed off-target editing. The main protocol modifications needed to achieve such high efficiencies were the addition of the DNA-PK inhibitor AZD7648, and the inclusion of spacer-breaking silent mutations in the donor in addition to mutations disrupting the PAM sequence. Critically, editing was even across the progenitor hierarchy, did not substantially distort the hierarchy or affect lineage outputs in colony-forming cell assays or the frequency of high self-renewal potential long-term culture initiating cells. 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 donors. 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.

Funder

Cancer Research Society

Terry Fox Research Institute

Fonds de Recherche du Québec - Santé

Publisher

eLife Sciences Publications, Ltd

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