Genome editing via homologous recombination (HR) (gene targeting) in human hematopoietic stem cells (HSCs) has the power to reveal gene-function relationships and potentially transform curative hematological gene and cell therapies. However, there are no comprehensive and reproducible protocols for targeting HSCs for HR. Herein, we provide a detailed protocol for the production, enrichment, and in vitro and in vivo analyses of HR-targeted HSCs by combining CRISPR/Cas9 technology with the use of rAAV6 and flow cytometry. Using this protocol, researchers can introduce single-nucleotide changes into the genome or longer gene cassettes with the precision of genome editing. Along with our troubleshooting and optimization guidelines, researchers can use this protocol to streamline HSC genome editing at any locus of interest. The in vitro HSC-targeting protocol and analyses can be completed in 3 weeks, and the long-term in vivo HSC engraftment analyses in immunodeficient mice can be achieved in 16 weeks. This protocol enables manipulation of genes for investigation of gene functions during hematopoiesis, as well as for the correction of genetic mutations in HSC transplantation-based therapies for diseases such as sickle cell disease, β-thalassemia, and primary immunodeficiencies.

中文翻译：

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人类造血干细胞中的CRISPR / Cas9基因组编辑。

在人类造血干细胞（HSC）中通过同源重组（HR）（基因靶向）进行基因组编辑具有揭示基因功能关系并潜在地转变治愈性血液学基因和细胞疗法的能力。但是，没有针对HR的针对HSC的全面且可复制的协议。本文中，我们通过结合CRISPR / Cas9技术与rAAV6和流式细胞仪的使用，为针对HR的HSC的产生，富集以及体外和体内分析提供了详细的协议。使用此协议，研究人员可以将单核苷酸变化引入基因组或更长的基因盒中，从而实现基因组编辑的精确性。连同我们的故障排除和优化指南，研究人员可以使用此协议在任何感兴趣的位点简化HSC基因组编辑。体外HSC靶向方案和分析可在3周内完成，免疫缺陷小鼠的体内长期HSC植入分析可在16周内完成。该协议能够操纵基因以研究造血过程中的基因功能，以及纠正基于HSC移植的镰状细胞病，β地中海贫血和原发性免疫缺陷等疾病的基因突变。