CRISPR-mediated Correction of Oncogenic AS-NMD in Splicing Factor Mutant Cancer

Area of Science:

• Molecular Biology
• Gene Regulation
• Cancer Therapeutics

Background:

• Alternative splicing coupled to nonsense-mediated mRNA decay (AS-NMD) is a critical gene expression regulator implicated in diseases like cancer.
• Poison exons (PEs) are conserved alternative exons triggering AS-NMD, often dysregulated in cancer to control tumor suppressor or oncoprotein levels.

Purpose of the Study:

• To investigate a CRISPR-based therapeutic strategy for manipulating PE-mediated oncogenic AS-NMD.
• To demonstrate proof-of-concept for targeting PEs in cancer treatment.

Main Methods:

• Utilized a CRISPR-based approach with paired guide RNA to delete a PE from the EZH2 gene in SRSF2-mutated leukemia cells.
• Analyzed the resulting EZH2 mRNA for PE absence, AS-NMD escape, and protein restoration.
• Assessed the functional consequences, including H3K27 histone methylation and chromatin regulation in hematopoietic stem cells.

Main Results:

• Successful deletion of the EZH2 PE using CRISPR editing.
• Restored EZH2 protein expression due to escaped AS-NMD.
• Reinstated H3K27 methylation and rescued defective chromatin regulation and hematopoietic stem cell differentiation.
• Demonstrated advantages of CRISPR over previously developed antisense technology.

Conclusions:

• CRISPR-mediated PE deletion is a viable therapeutic strategy for cancer and other diseases.
• This approach effectively targets oncogenic AS-NMD pathways by correcting gene expression.
• The study highlights CRISPR's potential for treating conditions driven by PE dysregulation.