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CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy
Published on: March 12, 2018
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Complex HBB gene editing outcomes revealed by a fluorescent reporter cell model
Cecile L Karsenty1, Daniel Betancourth2, Mingming Cao2
1Division Hematology/Oncology, Department of Pediatrics, Baylor College of Medicine, Cancer and Hematology Center, Texas Children's Hospital, Houston, TX 77030, USA.
Molecular Therapy. Nucleic Acids
|February 25, 2026
Summary
CRISPR-Cas9 gene editing for sickle cell disease shows varied outcomes. A new reporter model accurately tracks gene editing, improving safety assessments for novel therapies.
Area of Science:
- Molecular Biology
- Gene Editing Technologies
- Hematology
Background:
- CRISPR-Cas9 gene editing holds promise for treating sickle cell disease by correcting the HBB gene mutation.
- However, Cas9 editing at HBB can lead to unintended outcomes like indels, large deletions, and loss of allele events, raising safety concerns.
- Pharmacological modulators increasing homology-directed repair (HDR) may also elevate risks of large gene modifications.
Purpose of the Study:
- To develop a live-cell reporter model for precise, allele-specific monitoring of HBB gene editing outcomes.
- To enable high-throughput genotype-phenotype mapping and assess the safety of CRISPR/Cas9-based therapies.
Main Methods:
- Engineered a dual-fluorescent reporter cell model using GFP and BFP tagging for allele-specific HBB expression monitoring.
- Utilized fluorescence intensities to discriminate between in-frame alleles, frameshift indels, large deletions, and loss of allele events.
- Applied HDR-enhancing agents to test the reporter's sensitivity in capturing shifts in editing outcomes, including those missed by bulk assays.
Main Results:
- The reporter model successfully discriminated various HBB gene editing outcomes, including in-frame alleles, indels, large deletions, and loss of allele events.
- The model demonstrated high sensitivity in detecting shifts in loss of allele outcomes when HDR-enhancing agents were applied, outperforming bulk assays.
- Established a quantitative link between editing genotypes and erythroid phenotypes.
Conclusions:
- The developed live-cell reporter system is a valuable tool for dissecting gene editing outcomes influenced by different DNA repair pathways.
- This model facilitates the quantitative evaluation of CRISPR/Cas9 editing genotypes and their corresponding erythroid phenotypes.
- Provides a crucial platform for assessing the safety and efficacy of CRISPR/Cas9-based therapeutic strategies for sickle cell disease.
