Poster Presentation 47th Lorne Genome Conference 2026

Investigating the causality of genetic variants driving splicing changes using CRISPR-Cas9 base-editing (133484)

Michael Gitonobel 1 2 , Kai Wei Tan 1 , Aaryan Chhabra 1 , Aiswarya Balakrishnan 1 , Tamara Tongoi 2 , Sefi Rosenbluh 2 , Sridevi Sureshkumar 1 , Sureshkumar Balasubramanian 1
  1. School of Biological Sciences, Monash University, Clayton, VICTORIA, Australia
  2. Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Melbourne, Victoria, Australia

Splicing is an essential step in pre-mRNA processing in which introns are removed and exons are joined together to form mature mRNA. The usage of different splice sites will generate various mRNA transcripts from the same gene, which can either give rise to different protein isoforms or trigger nonsense-mediated mRNA decay to regulate gene expression. Genetic mutations may alter the usage of splice sites, which creates defective proteins and/or causes improper gene expression, resulting in disorders. Explorations to understand how genetic variations influence splicing pattern is just beginning to yield interesting results1, but there is still much more to be understood. In this investigation, we mapped thousands of genomic variants associated with the usage variation in specific splice sites across the human heart and testis tissues. We then used the CRISPR-Cas9 base-editing technology to assess whether these variants were causal in driving splice-site usage changes in HEK293T cells (which showed similar splicing patterns as seen in the tested tissues). To do this, we first meticulously chose representative single nucleotide polymorphisms (SNPs) to be edited using a series of strict parameters and then designed and cloned the sgRNAs for each chosen SNP into plasmids expressing CBE3.9max and ABE8e base-editors (which catalyse the C>T and A>G transition mutation, respectively). We transfected these plasmids into HEK293T cells and successfully edited 9 SNPs associated with the usage variation of 9 splice sites. We subsequently performed RNA extractions and compared the splicing patterns between transfected and non-transfected cells using RT-PCR. We established that 7 out of the 9 edited targets did cause alterations to splice-site usage. This work presents one of the first examples where CRISPR-Cas9 base-editing is used to successfully test the causality of genetic variants associated with molecular phenotypes and establishes CRISPR-Cas9 base-editing as a reliable method for demonstrating causations in eQTL/sQTL studies.

  1. 1) Dent et al. (2025) A basic framework to explain splice-site choice in eukaryotes. Nature Communications 16(8284), doi: 10.1038/s41467-025-63622-9