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

Methods Mol Biol. 2022;2429:281-306. doi: 10.1007/978-1-0716-1979-7_19.


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.

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

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems* / genetics
  • Gene Editing / methods
  • Hematopoietic Stem Cells / metabolism
  • High-Throughput Nucleotide Sequencing
  • RNA, Guide, Kinetoplastida* / genetics
  • RNA, Guide, Kinetoplastida* / metabolism


  • RNA, Guide