The CRISPR/Cas9 system has been used for genome editing of human and mouse cells. In this study, we established a protocol for gene knockout (KO) in mouse hematopoietic stem cells (HSCs). HSCs were highly purified from the bone marrow of tamoxifen-treated Cas9-EGFP/Cre-ER transgenic mice, maintained in serum-free polyvinyl alcohol culture with cytokines, lentivirally transduced with sgRNA-Crimson, and transplanted into lethally irradiated mice with competitor cells. Previous studies of Pax5 KO mice have shown B cell differentiation block. To verify our KO HSC strategy, we deleted Pax5 gene in 600 CD201⁺CD150⁺CD48⁻c-Kit⁺Sca-1⁺Lin⁻ cells (HSC1 cells), highly enriched in myeloid-biased HSCs, and CD201⁺CD150⁻CD48⁻ c-Kit⁺Sca-1⁺Lin⁻ cells (HSC2 cells), highly enriched in lymphoid-biased HSCs. As predicted, both Pax5 KO HSC1 and HSC2 cells showed few B cells in the peripheral blood and the accumulation of pro-B cells in the bone marrow of recipient mice. Our data suggesetd that myeloid-biased and lymphoid-biased HSCs share a common B cell differentiation pathway. This population-specific KO strategy will find its applications for gene editing in a varity of somatic cells, particuarly rare stem and progenitor cells from different tissues.

CRISPR/Cas9 系统已用于人类和小鼠细胞的基因组编辑。在这项研究中，我们建立了小鼠造血干细胞 (HSC) 基因敲除 (KO) 的方案。HSCs 从他莫昔芬处理的 Cas9-EGFP/Cre-ER 转基因小鼠的骨髓中高度纯化，保持在含有细胞因子的无血清聚乙烯醇培养物中，用 sgRNA-Crimson 慢病毒转导，并移植到具有竞争细胞的致死辐射小鼠中。先前对Pax5 KO 小鼠的研究显示 B 细胞分化阻滞。为了验证我们的 KO HSC 策略，我们删除了 600 CD201 ⁺ CD150 ⁺ CD48 ⁻ c-Kit ⁺ Sca-1 ⁺ Lin ^{− 中的}Pax5基因细胞（HSC1 细胞），高度富含骨髓偏向 HSC，CD201 ⁺ CD150 ^- CD48 ^- c-Kit ⁺ Sca-1 ⁺ Lin ^-细胞（HSC2 细胞），高度富含淋巴偏向 HSC。正如预测的那样，Pax5 KO HSC1 和 HSC2 细胞在外周血中显示出很少的 B 细胞，而在受体小鼠的骨髓中积聚了 pro-B 细胞。我们的数据表明，骨髓偏向和淋巴偏向的 HSC 共享一个共同的 B 细胞分化途径。这种特定于群体的 KO 策略将在各种体细胞，特别是来自不同组织的罕见干细胞和祖细胞中发现其基因编辑的应用。