Identification and Validation of CRISPR/Cas9 Off-Target Activity in Hematopoietic Stem and Progenitor Cells

Research output: Chapter in Book/Report/Conference proceedingsChapterpeer-review

Abstract

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.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages281-306
Number of pages26
DOIs
Publication statusPublished - 2022

Publication series

NameMethods in Molecular Biology
Volume2429
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • CRISPR/Cas9
  • Genome editing
  • GUIDE-seq
  • Hematopoietic stem and progenitor cells (HSPCs)
  • Next-generation sequencing (NGS)
  • Off-target

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