Related Experiment Video
Updated: Sep 24, 2025
Evaluation of Abnormal Growth-related Genes of Hematopoietic Stem and Progenitor Cells by Combining CRISPR/Cas9 Technology with Cell Counting
Published on: May 2, 2025
Identification and Validation of CRISPR/Cas9 Off-Target Activity in Hematopoietic Stem and Progenitor Cells
So Hyun Park1, Ciaran M Lee2, Gang Bao3
1Department of Bioengineering, Rice University, Houston, TX, USA.
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.
Insights
CRISPR/Cas9 genome editing shows promise for curing blood diseases in hematopoietic stem cells. This chapter details methods to identify and validate CRISPR/Cas9 off-target effects, ensuring safer gene therapy applications.
Area of Science:
- Molecular Biology
- Gene Therapy
- Hematology
Background:
- CRISPR/Cas9 technology offers potential for curing hematologic diseases through genome editing in hematopoietic stem and progenitor cells (HSPCs).
- Off-target mutations are a significant concern, potentially limiting the therapeutic application of CRISPR/Cas9 systems.
- Accurate identification and validation of these off-target effects are crucial for clinical translation.
Purpose of the Study:
- To provide a comprehensive protocol for identifying and validating CRISPR/Cas9 off-target effects specifically in HSPCs.
- To ensure the safety and efficacy of genome editing strategies for hematologic disorders.
Main Methods:
- Computational prediction of potential off-target sites.
- Experimental identification of off-target mutations in HSPCs.
- Quantification of off-target activity using next-generation sequencing (NGS).
Main Results:
- The described protocol enables robust identification and validation of CRISPR/Cas9 off-target sites.
- The method is adaptable to various CRISPR/Cas9 systems and cell types.
- Accurate assessment of off-target activity is achievable.
Conclusions:
- This chapter presents a validated methodology for assessing CRISPR/Cas9 off-target effects in HSPCs.
- The protocol supports the safe development of genome editing-based therapies for hematologic diseases.
- Standardized off-target analysis is essential for advancing CRISPR/Cas9 clinical applications.
