Oral Presentation 45th Lorne Genome Conference 2024

Molecular principles of CRISPR-Cas13 mismatch intolerance enable selective silencing of point-mutated oncogenic RNA with single-base precision (#29)

Carolyn E Shembrey 1 2 , Ray Yang 1 2 , Joshua ML Casan 1 2 , Wenxin Hu 1 2 , Teresa Sadras 1 2 , Krishneel Prasad 3 , Jake Shortt 1 2 , Ricky W Johnstone 1 2 , Joseph A Trapani 1 2 , Paul PG Ekert 1 4 , Mohamed Fareh 1 2
  1. Peter MacCallum Cancer Centre, Melbourne, VIC, Australia
  2. Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, VIC, Australia
  3. The University of Melbourne, Melbourne, VIC, Australia
  4. Personalised Medicine Program, Children's Cancer Institute, Randwick, NSW, Australia

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, most SNVs remain clinically unactionable due to the paucity of targeted small molecule inhibitors.

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 performed 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 with minimal off-target silencing of wildtype KRAS. We applied these principles to effectively silence oncogenic BRAF V600E and NRAS G12D, underscoring the adaptability of this platform to target various SNVs.

This proof-of-concept study (Shembrey et al., 2023, under review) demonstrates 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.

  1. Shembrey, C, ... , Ekert PG & Fareh M. Molecular principles of CRISPR-Cas13 mismatch intolerance enable selective silencing of point-mutated oncogenic RNA with single-base precision. bioRxiv 2023.09.26.557083 (2023) doi:10.1101/2023.09.26.557083.