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

Cell Rep. 2020 Sep 1;32(9):108093. doi: 10.1016/j.celrep.2020.108093.


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.

Keywords: CRISPR; adult hematopoietic stem cells; cell cycle; gene editing; gene therapy; homology-directed repair; quiescence.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems / genetics*
  • GATA3 Transcription Factor / metabolism*
  • Gene Editing / methods*
  • Genetic Therapy / methods*
  • Hematopoietic Stem Cells / metabolism*
  • Humans
  • Recombinational DNA Repair / genetics*
  • Stem Cells / metabolism*


  • GATA3 Transcription Factor
  • GATA3 protein, human