Poster Presentation 35th Lorne Cancer Conference 2023

Systematic silencing of point-mutated oncogenic transcripts with single-base precision design of CRISPR-Cas13 (#320)

Carolyn Shembrey 1 2 3 , Ray Yang 1 2 4 , Krishneel Prasad 1 2 4 , Wenxin Hu 1 2 4 , Michelle Haber 3 , Joseph A Trapani 1 2 , Paul G Ekert 1 2 3 5 , Mohamed Fareh 1 2
  1. Peter MacCallum Cancer Centre, Melbourne, VIC, Australia
  2. Rosie Lew Program in Immunotherapy and Cancer Cell Death Laboratory, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia
  3. Personalised Medicine Program, Children's Cancer Institute, Randwick, NSW, Australia
  4. The University of Melbourne, Melbourne, VIC, Australia
  5. Murdoch Children's Research Institute, Royal Children's Hospital, Parkville, Victoria, Australia

Comprehensive molecular profiling initiatives such as the Zero Childhood Cancers (ZCC) program have defined the enormous landscape of Single Nucleotide Variants (SNVs) present in human cancers. Cancer-associated SNVs are considered an attractive therapeutic target 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 permanent 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, the target binding process of Cas13 requires the recognition of a 30-nucleotide long RNA sequence, which endows this enzyme with extremely high specificity compared to the classical SpCas9 or eukaryotic RNA interference.

Here, we questioned whether Cas13 can be reprogrammed to suppress various SNV-containing transcripts such as those encoding BRAFV600E. Exclusive targeting of SNVs presents a significant challenge as these mutated transcripts differ from their wildtype counterparts by only one nucleotide, thus requiring extremely specific CRISPR RNAs (crRNAs) capable of discriminating between the two transcript variants with single-nucleotide precision. To identify such crRNAs, we performed comprehensive mutagenesis analysis of target-spacer interaction at single-nucleotide resolution whereby candidate crRNAs were serially mutated to determine their mismatch tolerance threshold. This screen revealed optimal crRNA design with enhanced transcriptional repression of mutant transcripts relative to their matched wildtype controls, which was later validated in various tumour models.

This proof-of-concept study demonstrates that the CRISPR-Cas13 system can be reprogrammed to target mutant transcripts with unprecedented accuracy, revealing the enormous potential for this tool in personalised transcriptome editing.