Principles of CRISPR-Cas13 mismatch intolerance enable selective silencing of point-mutated oncogenic RNA with single-base precision
- Author(s)
- Shembrey, C; Yang, R; Casan, J; Hu, W; Chen, H; Singh, GJ; Sadras, T; Prasad, K; Shortt, J; Johnstone, RW; Trapani, JA; Ekert, PG; Fareh, M;
- Details
- Publication Year 2024-12-20,Volume 10,Issue #51,Page eadl0731
- Journal Title
- Science Advances
- Publication Type
- Research article
- 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.
- Publisher
- American Association for the Advancement of Science
- Keywords
- *CRISPR-Cas Systems; Humans; RNA, Guide, CRISPR-Cas Systems/genetics; Point Mutation; Gene Silencing; Gene Editing/methods; Polymorphism, Single Nucleotide; Proto-Oncogene Proteins p21(ras)/genetics; Base Pair Mismatch; Membrane Proteins/genetics; Oncogenes; RNA, Neoplasm/genetics/metabolism; GTP Phosphohydrolases; Proto-Oncogene Proteins B-raf
- Department(s)
- Laboratory Research
- Publisher's Version
- https://doi.org/10.1126/sciadv.adl0731
- Open Access at Publisher's Site
https://doi.org/10.1126/sciadv.adl0731- Terms of Use/Rights Notice
- Refer to copyright notice on published article.
Creation Date: 2025-01-23 02:18:54
Last Modified: 2025-01-23 02:30:24