Poster Presentation 43rd Lorne Genome Conference 2022

Towards Cas13-mediated transcriptional repression of tumour drivers with single-nucleotide precision (#211)

Carolyn E Shembrey 1 2 3 , Krishneel Prasad 1 2 , Wenxin Hu 1 2 , Michelle Haber 3 , Paul G Ekert 1 2 3 , Joseph A Trapani 1 2 , Mohamed Fareh 1 2
  1. Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, 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

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, 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. Indeed, we and others have recently reprogrammed Cas13 tools to suppress multiple replication-competent SARS-CoV-2 variants with high specificity1.

Here, we questioned whether Cas13 can be reprogrammed to suppress various tumour driver transcripts. Among these drivers, we are particularly interested in silencing single-nucleotide variants (SNVs) that drive multiple cancers including melanoma, colorectal cancer and neuroblastoma. 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. In order 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.

 

 

 

  1. 1 Fareh, M., Zhao, W., Hu, W. et al. Reprogrammed CRISPR-Cas13b suppresses SARS-CoV-2 replication and circumvents its mutational escape through mismatch tolerance. Nat Commun 12, 4270 (2021). https://doi.org/10.1038/s41467-021-24577-9