人类造血干细胞和祖细胞中CRISPR-Cas9基因编辑过程中体外激活培养条件优化方案

IF 1.3 Q4 BIOCHEMICAL RESEARCH METHODS

STAR Protocols Pub Date : 2025-06-20 Epub Date: 2025-03-31 DOI:10.1016/j.xpro.2025.103722

Lucrezia Della Volpe, Roberta Vacca, Raffaella Di Micco

{"title":"人类造血干细胞和祖细胞中CRISPR-Cas9基因编辑过程中体外激活培养条件优化方案","authors":"Lucrezia Della Volpe, Roberta Vacca, Raffaella Di Micco","doi":"10.1016/j.xpro.2025.103722","DOIUrl":null,"url":null,"abstract":"Long-range correction strategies require ex vivo activation of hematopoietic stem and progenitor cells (HSPCs) to engage the homology-directed repair (HDR) mechanism, but prolonged culture causes harmful cellular responses, reducing the long-term functionality of gene-edited (GE) HSPCs. Here, we present a protocol for optimizing culture conditions for ex vivo activation during CRISPR-Cas9 gene editing in human HSPCs. We describe steps for HSPC thawing, ex vivo treatments, gene editing, and downstream in vitro and in vivo analyses to assess the functionality of GE-HSPCs. For complete details on the use and execution of this protocol, please refer to della Volpe et al.1.","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103722"},"PeriodicalIF":1.3000,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11999203/pdf/","citationCount":"0","resultStr":"{\"title\":\"Protocol for optimizing culture conditions for ex vivo activation during CRISPR-Cas9 gene editing in human hematopoietic stem and progenitor cells.\",\"authors\":\"Lucrezia Della Volpe, Roberta Vacca, Raffaella Di Micco\",\"doi\":\"10.1016/j.xpro.2025.103722\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"Long-range correction strategies require ex vivo activation of hematopoietic stem and progenitor cells (HSPCs) to engage the homology-directed repair (HDR) mechanism, but prolonged culture causes harmful cellular responses, reducing the long-term functionality of gene-edited (GE) HSPCs. Here, we present a protocol for optimizing culture conditions for ex vivo activation during CRISPR-Cas9 gene editing in human HSPCs. We describe steps for HSPC thawing, ex vivo treatments, gene editing, and downstream in vitro and in vivo analyses to assess the functionality of GE-HSPCs. For complete details on the use and execution of this protocol, please refer to della Volpe et al.1.\",\"PeriodicalId\":34214,\"journal\":{\"name\":\"STAR Protocols\",\"volume\":\"6 2\",\"pages\":\"103722\"},\"PeriodicalIF\":1.3000,\"publicationDate\":\"2025-06-20\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11999203/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"STAR Protocols\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.1016/j.xpro.2025.103722\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2025/3/31 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"Q4\",\"JCRName\":\"BIOCHEMICAL RESEARCH METHODS\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"STAR Protocols","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1016/j.xpro.2025.103722","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2025/3/31 0:00:00","PubModel":"Epub","JCR":"Q4","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}

引用次数: 0

摘要

长期纠正策略需要体外激活造血干细胞和祖细胞（HSPCs）来参与同源定向修复（HDR）机制，但长时间培养会导致有害的细胞反应，降低基因编辑（GE） HSPCs的长期功能。在这里，我们提出了一种优化培养条件的方案，以便在人造血干细胞的CRISPR-Cas9基因编辑过程中体外激活。我们描述了HSPC解冻、离体处理、基因编辑和下游体外和体内分析的步骤，以评估ge -HSPC的功能。有关本协议使用和执行的完整细节，请参阅della Volpe等人1。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

查看原文本刊更多论文

Protocol for optimizing culture conditions for ex vivo activation during CRISPR-Cas9 gene editing in human hematopoietic stem and progenitor cells.

Long-range correction strategies require ex vivo activation of hematopoietic stem and progenitor cells (HSPCs) to engage the homology-directed repair (HDR) mechanism, but prolonged culture causes harmful cellular responses, reducing the long-term functionality of gene-edited (GE) HSPCs. Here, we present a protocol for optimizing culture conditions for ex vivo activation during CRISPR-Cas9 gene editing in human HSPCs. We describe steps for HSPC thawing, ex vivo treatments, gene editing, and downstream in vitro and in vivo analyses to assess the functionality of GE-HSPCs. For complete details on the use and execution of this protocol, please refer to della Volpe et al.¹.

求助全文

通过发布文献求助，成功后即可免费获取论文全文。去求助

来源期刊