Single nucleotide variants (SNVs) are extremely prevalent in human cancers. For instance, KRAS point mutations at codon 12 occur in approximately 90% of pancreatic cancers. Cancer-associated SNVs are considered attractive therapeutic targets due to their restricted expression in tumour cells. However, the majority of SNVs remain undruggable, and traditional methods of protein-targeting drug design are expensive, time-consuming, and low-yield.
CRISPR-Cas9 molecular tools have revolutionised our ability to perform targeted genome editing. However, recent reports have linked the on- and off-target nuclease activity of CRISPR-Cas9 to chromosomal loss and chromatin rearrangement, which unfortunately limit the therapeutic potential of this tool. Conversely, CRISPR-Cas13 is an RNA-guided RNA-targeting nuclease that enables precise and efficient cleavage of single-stranded RNA without altering genomic DNA. Moreover, due to its longer guide RNA, Cas13 has extremely high specificity as compared with classical SpCas9 or eukaryotic RNA interference. Although CRISPR-Cas13 has been deployed to specifically target RNAs such as overexpressed oncogenes and fusion transcripts, silencing 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 BRAF V600E as well as NRAS G12D, for which there are no existing small molecular inhibitors, underscoring the adaptability of this platform to silence various “undruggable” SNVs.
This proof-of-concept study (Shembrey et al., 2023, under review) demonstrates a highly precise RNA targeting strategy that effectively suppresses single-nucleotide variants whilst sparing wildtype transcripts. This tool may be of great value for elucidating the function of point-mutated variants of unknown significance or, with further development, to therapeutically suppress oncogenic drivers with single-nucleotide precision.