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Published on: September 1, 2019
Systematic screening for functional exon-skipping isoforms using the CRISPR-RfxCas13d system
Qiang Sun1, Xuejie Ma2, Qianqian Ning3
1Cancer Institute, Xuzhou Medical University, Xuzhou 221004, Jiangsu, China; Center of Clinical Oncology, the Affiliated Hospital of Xuzhou Medical University, Xuzhou 221004, Jiangsu, China; Jiangsu Center for the Collaboration and Innovation of Cancer Biotherapy, Cancer Institute, Xuzhou Medical University, Xuzhou 221004, Jiangsu, China; Center for RNA Medicine, the Fourth Affiliated Hospital of School of Medicine, International School of Medicine, International Institutes of Medicine, Zhejiang University, Yiwu 320000, Zhejiang, China.
Abstract:
Exon skipping (ES) is the most prevalent form of alternative splicing and a hallmark of tumorigenesis, yet its functional roles remain underexplored. Here, we present a CRISPR-RfxCas13d-based platform for transcript-specific silencing of ES-derived isoforms using guide RNAs (gRNAs) targeting exon-exon junctions. We designed a transcriptome-wide gRNA library against 3,744 human ES events and conducted loss-of-function screens in colorectal cancer (CRC) cells in vitro and in vivo. This screen uncovered multiple ES events essential for CRC growth, notably HMGN3 Δ6, an isoform arising from exon 6 skipping, which enhanced tumor proliferation. Functional validation confirmed the oncogenic role of HMGN3 Δ6 and its necessity for CRC progression. Our study establishes CRISPR-RfxCas13d as a powerful tool for isoform-specific functional genomics and reveals a widespread, previously uncharacterized layer of tumor biology driven by ES. These findings position ES-derived transcripts as promising targets for therapeutic intervention in cancer.
Insights
This study introduces a new CRISPR tool to silence specific cancer-driving gene variants. Researchers identified exon skipping events, like HMGN3 Δ6, crucial for colorectal cancer growth and progression.
Area of Science:
- Molecular Biology
- Genomics
- Cancer Research
Background:
- Exon skipping (ES) is a common splicing alteration in cancer, but its functional impact is poorly understood.
- Understanding ES-derived isoforms is crucial for cancer biology and therapeutic development.
Purpose of the Study:
- To develop a transcript-specific gene silencing platform for studying ES events.
- To identify functional ES events driving colorectal cancer (CRC) progression.
Main Methods:
- Developed a CRISPR-RfxCas13d system for targeting exon-exon junctions.
- Created a transcriptome-wide guide RNA library for 3,744 human ES events.
- Performed loss-of-function screens in CRC cells (in vitro and in vivo).
Main Results:
- Identified multiple ES events critical for CRC cell growth.
- Discovered HMGN3 Δ6, an ES isoform, significantly enhances tumor proliferation.
- Validated the oncogenic role of HMGN3 Δ6 in CRC progression.
Conclusions:
- CRISPR-RfxCas13d is effective for isoform-specific functional genomics.
- ES-derived transcripts represent a significant, understudied layer of tumor biology.
- ES events, like HMGN3 Δ6, are potential therapeutic targets in cancer.
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