Sharma Devesh, Sinha Roshani, Lesch Benjamin J, Cromer M Kyle
Department of Surgery, University of California, San Francisco, San Francisco, CA 94143, USA; Eli & Edythe Broad Center for Regeneration Medicine, University of California, San Francisco, San Francisco, CA 94143, USA.
Department of Surgery, University of California, San Francisco, San Francisco, CA 94143, USA; Eli & Edythe Broad Center for Regeneration Medicine, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Bioengineering & Therapeutic Sciences, University of California, San Francisco, San Francisco, CA 94158, USA.
STAR Protoc. 2025 Aug 5;6(3):104018. doi: 10.1016/j.xpro.2025.104018.
Here, we present a protocol for genome editing in human hematopoietic stem and progenitor cells (HSPCs) using CRISPR-Cas9 ribonucleoproteins and adeno-associated virus (AAV)-mediated homology-directed repair. We describe steps for AAV production, purification, and titration; HSPC thawing and culture; genome editing; and quantification of editing frequencies. We then detail procedures for erythroid differentiation assays. This protocol ensures high editing efficiency while maintaining cell viability and engraftment potential. For complete details on the use and execution of this protocol, please refer to Chu et al..
在此，我们展示了一种使用CRISPR-Cas9核糖核蛋白和腺相关病毒（AAV）介导的同源定向修复在人类造血干细胞和祖细胞（HSPCs）中进行基因组编辑的方案。我们描述了AAV生产、纯化和滴定的步骤；HSPC解冻和培养；基因组编辑；以及编辑频率的量化。然后，我们详细介绍了红系分化测定的程序。该方案确保了高编辑效率，同时保持细胞活力和植入潜力。有关该方案的使用和执行的完整详细信息，请参考Chu等人的研究。
