Molecular principles of CRISPR-Cas13 mismatch intolerance enable selective silencing of point-mutated oncogenic RNA with single-base precision

Single nucleotide variants (SNVs) are extremely prevalent in human cancers. For instance, KRAS mutations occur in over 90% of pancreatic cancers and ~40% of colorectal cancers. Virtually all KRAS mutations are SNVs, most of which remain clinically unactionable. The programmable RNA nuclease CRISPR-Cas13 has been deployed to specifically target RNAs such as overexpressed oncogenes and fusion transcripts. However, silencing oncogenic SNVs with single-base precision remains extremely challenging due to the intrinsic mismatch tolerance of Cas13. Here, we developed a comprehensive mutagenesis analysis of target-spacer interactions at single-nucleotide resolution, which revealed key spacer nucleotide positions intolerant to mismatches. We show that introducing synthetic mismatches at these precise positions enables de novo design of CRISPR RNA (crRNA) with strong preferential silencing of SNV transcripts. We demonstrate that our top-performing crRNAs possess prominent SNV-selectivity with dose-dependent silencing activity against all KRAS G12 variants at both the RNA and protein levels with minimal off-target silencing of wildtype KRAS. We applied these design principles to effectively silence oncogenic NRAS G12D and BRAF V600E transcripts, underscoring the adaptability of this platform to silence various SNVs. 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. ### Competing Interest Statement The findings in this study are covered by a patent deposited by the Peter MacCallum Cancer Centre. The authors declare no other conflict of interest.