Department of Pediatrics, Stanford University, Stanford, California, USA.
Nat Protoc. 2018 Feb;13(2):358-376. doi: 10.1038/nprot.2017.143. Epub 2018 Jan 25.
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.
通过同源重组（HR）（基因靶向）对人类造血干细胞（HSCs）进行基因组编辑，具有揭示基因功能关系的能力，并有可能改变治疗性血液基因和细胞疗法。然而，目前尚无针对 HR 靶向 HSCs 的全面和可重复的方案。在此，我们提供了一种详细的方案，该方案结合了 CRISPR/Cas9 技术和 rAAV6 的使用以及流式细胞术，用于产生、富集以及体外和体内分析 HR 靶向 HSCs。使用此方案，研究人员可以利用基因组编辑的精度将单核苷酸变化或更长的基因盒引入基因组中。结合我们的故障排除和优化指南，研究人员可以使用此方案简化任何感兴趣的 HSC 基因组编辑。体外 HSC 靶向方案和分析可以在 3 周内完成，而免疫缺陷小鼠中的长期体内 HSC 植入分析可以在 16 周内完成。该方案可用于操纵基因，以研究造血过程中的基因功能，以及校正基于 HSC 移植的疗法中的遗传突变，如镰状细胞病、β-地中海贫血和原发性免疫缺陷。
