Oral Presentation 46th Lorne Genome Conference 2025

Disease mechanisms resolved by transactivation of silent genes (115026)

Emmylou Nicolas-Martinez 1 2 , Christian Pflueger 3 4 , Alison Gardner 1 2 , Mark A Corbett 1 2 , Tarin Ritchie 1 2 , The PERSYST Investigator Team 5 , Ryan Lister 3 4 , Jozef Gecz 1 2 , Lachlan A Jolly 1 2
  1. University of Adelaide, Adelaide, AUSTRALIA, Australia
  2. Robinson Research Institute, The University of Adelaide, Adelaide, S.A., Australia
  3. Harry Perkins Institute of Medical Research, Perth, W.A., Australia
  4. The University of Western Australia, Perth, W.A., Australia
  5. National Collaboration, Australia

Understanding the impact that genetic variants suspected to cause disease have on RNA splicing is crucial for both diagnosis and precision medicine opportunities including antisense oligonucleotide therapies. This is enabled through RNA studies involving transcriptomics followed by targeted assays using RNA isolated from clinically accessible tissues (CATs) such as the patients’ blood or skin. Insufficient disease gene expression in CATs does however pose a major barrier to RNA based investigations, which we show is relevant to 1,436 Mendelian disease genes, herein termed ‘silent Mendelian genes’ (SMGs). We pursued CRISPR-activation based gene transactivation in patient derived human dermal fibroblasts (HDFs) to overcome this limitation. We performed ATAC-seq of HDFs to identify open-chromatin regions in the promoters of SMGs to inform gRNA design. An initial single cell Perturb-seq transactivation screen of 40 SMGs in HDFs highlighted broad utility (25/40 SMGs transactivated in >100 cells), and an underlying relationship between the level of gRNA expression and transactivation. This stimulated our development of a four-plex gRNA transactivation system culminating in the 6- to 90,000-fold induction of expression of 41/41 (100%) SMGs tested individually in HDFs thus far. RNA-seq revealed that the magnitude (1-3000 transcripts per million) and isoform diversity of transactivated SMGs in HDFs was comparable to the expression of SMGs in their clinically relevant tissues. We applied SMG transactivation combined with short- and long-read nanopore RNA sequencing to resolve the splicing impact of 14 variants in SMGs, (e.g. USH2A, SCN1A, DMD), using HDFs derived from patients, resulting in new diagnosis, disease mechanisms and precision medicine opportunities. Transactivation represents a functional genomic solution to investigate the >88K variants of uncertain significance in SMGs captured in ClinVar to date. More broadly, the gene transactivation system enables robust functional genomic studies of non- or lowly-expressed genes.