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Updated: Mar 29, 2026
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
ASXL1 mutation correction by CRISPR/Cas9 restores gene function in leukemia cells and increases survival in mouse
Simona Valletta1, Hamid Dolatshad1, Matthias Bartenstein2
1Bloodwise Molecular Haematology Unit, Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford and BRC Blood Theme, NIHR Oxford Biomedical Centre, Oxford University Hospital, Oxford, UK.
Abstract:
Recurrent somatic mutations of the epigenetic modifier and tumor suppressor ASXL1 are common in myeloid malignancies, including chronic myeloid leukemia (CML), and are associated with poor clinical outcome. CRISPR/Cas9 has recently emerged as a powerful and versatile genome editing tool for genome engineering in various species. We have used the CRISPR/Cas9 system to correct the ASXL1 homozygous nonsense mutation present in the CML cell line KBM5, which lacks ASXL1 protein expression. CRISPR/Cas9-mediated ASXL1 homozygous correction resulted in protein re-expression with restored normal function, including down-regulation of Polycomb repressive complex 2 target genes. Significantly reduced cell growth and increased myeloid differentiation were observed in ASXL1 mutation-corrected cells, providing new insights into the role of ASXL1 in human myeloid cell differentiation. Mice xenografted with mutation-corrected KBM5 cells showed significantly longer survival than uncorrected xenografts. These results show that the sole correction of a driver mutation in leukemia cells increases survival in vivo in mice. This study provides proof-of-concept for driver gene mutation correction via CRISPR/Cas9 technology in human leukemia cells and presents a strategy to illuminate the impact of oncogenic mutations on cellular function and survival.
Insights
CRISPR/Cas9 gene editing corrected the ASXL1 mutation in chronic myeloid leukemia (CML) cells, restoring normal function and improving cell differentiation. This approach significantly increased survival in mice, demonstrating its therapeutic potential for leukemia.
Area of Science:
- Molecular Biology
- Genetics
- Cancer Research
Background:
- ASXL1 mutations are common in myeloid malignancies and linked to poor outcomes.
- CRISPR/Cas9 is a powerful tool for genome editing.
Purpose of the Study:
- To correct the ASXL1 mutation in chronic myeloid leukemia (CML) cells using CRISPR/Cas9.
- To investigate the functional impact of ASXL1 correction on leukemia cells and in vivo survival.
Main Methods:
- Utilized CRISPR/Cas9 to correct the ASXL1 homozygous nonsense mutation in the KBM5 CML cell line.
- Assessed ASXL1 protein re-expression, target gene regulation, cell growth, and myeloid differentiation.
- Evaluated survival in mice xenografted with corrected and uncorrected cells.
Main Results:
- CRISPR/Cas9 successfully corrected the ASXL1 mutation, leading to protein re-expression and restored function.
- Corrected cells showed reduced growth, increased myeloid differentiation, and down-regulation of Polycomb targets.
- Mice xenografted with corrected cells exhibited significantly longer survival.
Conclusions:
- Sole correction of the ASXL1 driver mutation in leukemia cells can improve survival in vivo.
- This study provides proof-of-concept for CRISPR/Cas9-mediated driver gene correction in human leukemia.
- The findings offer a strategy to study oncogenic mutation impact on cellular function and survival.
