Open AccessEfficient gene editing of human long-term hematopoietic stem cells validated by clonal tracking
Author(s) -
Samuele Ferrari,
Aurélien Jacob,
Stefano Beretta,
Giulia Unali,
Luisa Albano,
Valentina Vavassori,
Davide Cittaro,
Dejan Lazarevic,
Chiara Brombin,
Federica Cugnata,
Anna KajasteRudnitski,
Ivan Merelli,
Pietro Genovese,
Luigi Naldini
Publication year - 2020
Publication title -
nature biotechnology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 15.358
H-Index - 445
eISSN - 1546-1696
pISSN - 1087-0156
DOI - 10.1038/s41587-020-0551-y
Subject(s) - biology , stem cell , haematopoiesis , genome editing , cell cycle , hematopoietic stem cell , microbiology and biotechnology , gene , cancer research , genetics , crispr
Targeted gene editing in hematopoietic stem cells (HSCs) is a promising treatment for several diseases. However, the limited efficiency of homology-directed repair (HDR) in HSCs and the unknown impact of the procedure on clonal composition and dynamics of transplantation have hampered clinical translation. Here, we apply a barcoding strategy to clonal tracking of edited cells (BAR-Seq) and show that editing activates p53, which substantially shrinks the HSC clonal repertoire in hematochimeric mice, although engrafted edited clones preserve multilineage and self-renewing capacity. Transient p53 inhibition restored polyclonal graft composition. We increased HDR efficiency by forcing cell-cycle progression and upregulating components of the HDR machinery through transient expression of the adenovirus 5 E4orf6/7 protein, which recruits the cell-cycle controller E2F on its target genes. Combined E4orf6/7 expression and p53 inhibition resulted in HDR editing efficiencies of up to 50% in the long-term human graft, without perturbing repopulation and self-renewal of edited HSCs. This enhanced protocol should broaden applicability of HSC gene editing and pave its way to clinical translation.