Identification and Validation of CRISPR/Cas9 Off-Target Activity in Hematopoietic Stem and Progenitor Cells.
Identification and Validation of CRISPR/Cas9 Off-Target Activity in Hematopoietic Stem and Progenitor Cells.
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造血干细胞和祖细胞中 CRISPR/Cas9 脱靶活性的鉴定和验证。
DOI：
10.1007/978-1-0716-1979-7_19
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Bao,Gang
中科院分区：
文献类型：
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作者：
Park,SoHyun;Lee,CiaranM;Bao,Gang
使用CRISPR/Cas9在造血干细胞和祖细胞（HSPC）中进行靶向基因组编辑可能会为血液病提供永久性治疗。然而，CRISPR/Cas9系统用于治疗性基因组编辑的效用可能会受到其脱靶效应的影响。在本章中，我们概述了HSPC中CRISPR/Cas9脱靶鉴定和验证的程序。该方法广泛适用于不同的CRISPR/Cas9系统和细胞类型。使用该方案，研究人员可以对潜在的脱靶位点进行计算预测和实验鉴定，然后通过下一代测序进行脱靶活性定量。
Targeted genome editing in hematopoietic stem and progenitor cells (HSPCs) using CRISPR/Cas9 can potentially provide a permanent cure for hematologic diseases. However, the utility of CRISPR/Cas9 systems for therapeutic genome editing can be compromised by their off-target effects. In this chapter, we outline the procedures for CRISPR/Cas9 off-target identification and validation in HSPCs. This method is broadly applicable to diverse CRISPR/Cas9 systems and cell types. Using this protocol, researchers can perform computational prediction and experimental identification of potential off-target sites followed by off-target activity quantification by next-generation sequencing.