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Updated: Jun 4, 2025
Dual CRISPR-Interference Strategy for Targeting Synthetic Lethal Interactions Between Non-Coding RNAs in Cancer Cells
Published on: May 30, 2025
Principles of CRISPR-Cas13 mismatch intolerance enable selective silencing of point-mutated oncogenic RNA with
Carolyn Shembrey1,2, Ray Yang1,2, Joshua Casan1,2
1Rosie Lew Program in Immunotherapy and Cancer Cell Death Laboratory, Peter MacCallum Cancer Centre, Melbourne 3000, Australia.
Abstract:
Single-nucleotide variants (SNVs) are extremely prevalent in human cancers, although most of these remain clinically unactionable. The programmable RNA nuclease CRISPR-Cas13 has been deployed to specifically target oncogenic RNAs. However, silencing oncogenic SNVs with single-base precision remains extremely challenging due to the intrinsic mismatch tolerance of Cas13. Here, we show that introducing synthetic mismatches at precise positions of the spacer sequence enables de novo design of guide RNAs [CRISPR RNAs (crRNAs)] with strong preferential silencing of point-mutated transcripts. We applied these design principles to effectively silence the oncogenic KRAS G12 hotspot, NRAS G12D and BRAF V600E transcripts with minimal off-target silencing of the wild-type transcripts, underscoring the adaptability of this platform to silence various SNVs. Unexpectedly, the SNV-selective crRNAs harboring mismatched nucleotides reduce the promiscuous collateral activity of the RfxCas13d ortholog. These findings demonstrate that the CRISPR-Cas13 system can be reprogrammed to target mutant transcripts with single-base precision, showcasing the tremendous potential of this tool in personalized transcriptome editing.
Insights
Researchers developed novel CRISPR-Cas13 guide RNAs (crRNAs) to precisely silence cancer-driving single-nucleotide variants (SNVs) in RNA. This breakthrough enables targeted transcriptome editing with reduced off-target effects and collateral activity.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- Single-nucleotide variants (SNVs) are common in cancer but often clinically undruggable.
- CRISPR-Cas13 technology targets RNA but struggles with single-base precision due to mismatch tolerance.
Purpose of the Study:
- To engineer CRISPR-Cas13 guide RNAs (crRNAs) for precise silencing of oncogenic SNVs.
- To demonstrate the platform's adaptability for various cancer-related SNVs.
- To investigate the impact of modified crRNAs on Cas13 collateral activity.
Main Methods:
- Designed novel crRNAs with synthetic mismatches to enhance specificity for mutated transcripts.
- Applied the designed crRNAs to target specific oncogenic SNVs in KRAS, NRAS, and BRAF.
- Assessed the silencing efficiency and off-target effects on wild-type transcripts.
- Evaluated the collateral activity of the RfxCas13d ortholog with modified crRNAs.
Main Results:
- Achieved preferential silencing of oncogenic KRAS G12, NRAS G12D, and BRAF V600E transcripts.
- Demonstrated minimal off-target silencing of wild-type transcripts.
- Observed a reduction in collateral activity of RfxCas13d when using SNV-selective crRNAs.
Conclusions:
- CRISPR-Cas13 can be reprogrammed for single-base precision targeting of mutant transcripts.
- This platform shows significant potential for personalized transcriptome editing in cancer therapy.
- Engineered crRNAs offer a strategy to improve Cas13 specificity and reduce unwanted side effects.
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