Controlled Cycling and Quiescence Enables Efficient HDR in Engraftment-Enriched Adult Hematopoietic Stem and Progenitor Cells
Controlled Cycling and Quiescence Enables Efficient HDR in Engraftment-Enriched Adult Hematopoietic Stem and Progenitor Cells
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DOI：
10.1016/j.celrep.2020.108093
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发表时间：
2020-09-01
期刊：
影响因子：
8.8
通讯作者：
Corn, Jacob E.
中科院分区：
文献类型：
--
作者：
Shin, Jiyung J.;Schroeder, Markus S.;Corn, Jacob E.
基因组编辑通常采用通过非同源末端连接 (NHEJ) 进行易错序列破坏或通过同源定向修复 (HDR) 进行序列替换的形式。尽管 NHEJ 通常是有效的，但 HDR 在原代细胞中通常很困难。在这里，我们结合使用免疫表型分析、下一代测序和单细胞 RNA 测序来研究和重新编程成体造血干细胞和祖细胞亚群的基因组编辑结果。我们发现，虽然静止的干细胞富集细胞大多使用 NHEJ，但具有相同免疫表型的非静止细胞同时使用 NHEJ 和 HDR。编辑前诱导静止会导致所有细胞亚型的 HDR 丢失。我们开发了一种受控循环和静止的策略，使离体和体内静止干细胞的 HDR/NHEJ 比率增加 6 倍。我们的结果强调了编辑和细胞生理学之间的紧张关系，并提出了操纵静止细胞进行研究和治疗性基因组编辑的策略。
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.