Poster Presentation 47th Lorne Genome Conference 2026

Challenges in developing a novel screen to identify 3D genome architecture regulators (133242)

Wendy Jia 1 2 , Hannah D. Coughlan 1 3 , Oliver Ozaydin 1 2 , Jakob Schuster 1 2 , Mathew Chu 1 2 , Miles B. Horton 1 2 , WingFuk Chan 1 2 , Daniel V Brown 1 2 , Esther Bandala Sanchez 1 2 , Michael J.G. Milevskiy 1 2 , Sarahi Mendoza Rivera 1 2 , Jane E Visvader 1 2 , Matthew E Ritchie 1 2 , Rory Bowden 1 2 , Gordon K Smyth 1 2 3 , Stephin J Vervoort 1 2 , Shalin H Naik 1 2 , Tom S Weber 1 2 , Rhys S Allan 1 2 , Timothy M Johanson 1 2
  1. Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia
  2. The University of Melbourne Department of Medical Biology, The University of Melbourne, Parkville, Victoria, Australia
  3. School of Mathematics and Statistics, The University of Melbourne, Parkville, Victoria, Australia

The eukaryotic genome is a polymer structure organised in three dimensions (3D) to generate a hierarchical organisation that is crucial for cell function and identity. Despite efforts to characterise the molecular machinery driving this genome organisation, relatively few proteins have identified as universal genome organisers. To address this, we developed a CRISPR-based screen called 3CaPER (Chromatin Conformation Capture with Prime Editing and RNA knockdown) to uncover novel regulators of 3D genome organisation. This innovative screen couples Cas13d mediated knock-down (KD) of a panel of candidate genes with prime editing-driven cellular barcoding that can be read out using barcode enriched long read chromosome conformation capture (Capture Pore-C) to directly resolve the impact of target gene KD on genome organisation. As a proof-of-concept study, we cloned a sub-library containing 28 gene candidates, including known 3D genome regulators and apoptotic pathway genes to account for cell death in the screen read out. Analysis of the sub-library screen revealed technical limitations leading to a weak signal to noise ratio, and thus the inability to connect gene KD with loss of genome architecture. Our experience highlights both the challenges and opportunities for high-throughput approaches to discover novel regulators of genome organisation.