Controlled Cycling and Quiescence Enables Efficient HDR in Engraftment-Enriched Adult Hematopoietic Stem and Progenitor Cells

The Corn lab's paper on "Controlled Cycling and Quiescence Enables Efficient HDR in Engraftment-Enriched Adult Hematopoietic Stem and Progenitor Cells" has been published in Cell Reports.

Highlights

  • HSCs require more time to activate HDR pathways than do progenitor cells
  • Rapamycin and CHIR99021 in media induce quiescence and maintain stemness in HSPCs
  • Quiescent HSPCs perform only NHEJ, whereas cycling HSPCs perform both NHEJ and HDR
  • Inducing quiescence after a short period of cycling yields HDR in engrafting HSCs

Summary

Genome editing often takes the form of either error-prone sequence disruption by non-homologous end joining (NHEJ) or sequence replacement by homology-directed repair (HDR). Although NHEJ is generally effective, HDR is often difficult in primary cells. Here, we use a combination of immunophenotyping, next-generation sequencing, and single-cell RNA sequencing to investigate and reprogram genome editing outcomes in subpopulations of adult hematopoietic stem and progenitor cells. We find that although quiescent stem-enriched cells mostly use NHEJ, non-quiescent cells with the same immunophenotype use both NHEJ and HDR. Inducing quiescence before editing results in a loss of HDR in all cell subtypes. We develop a strategy of controlled cycling and quiescence that yields a 6-fold increase in the HDR/NHEJ ratio in quiescent stem cells ex vivo and in vivo. Our results highlight the tension between editing and cellular physiology and suggest strategies to manipulate quiescent cells for research and therapeutic genome editing.

Read the Publication in Cell Reports (Open Access)

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Graphical abstract, highlights & title from Shin J.J. et al. (2020) Cell Reports published under a CC BY 4.0 License