Abstract 3973: Precision RNAi using synthetic shRNAmir target sites

Abstract Genetic loss-of-function methods including short-hairpin RNAs (shRNAs) and CRISPR are key methods for target gene validation across a variety of different disease areas. While these methods have revolutionized target gene discovery and characterization, both methods suffer from limitations in the context of in vivo target validation due to off-target effects and insufficient knock-down for shRNAs and resistant clones for CRISPR. In this study, we describe a method, artificial RNA interference (ARTi) that overcomes these limitations by fundamentally changing the basic experimental strategy of RNAi-based loss-of-function studies. Instead of newly designing gene-specific shRNA for individual target genes, ARTi utilizes ultra-effective and selective artificial miRNA-based shRNAs that do not match any transcribed gene in the target genome. In addition, these sequences are optimized for ultra-efficient miRNA processing and knockdown of synthetic target genes, and are deeply characterized to not trigger major off-target effects, enabling highly stringent target validation in vivo with unprecedented temporal control, selectivity and potency. We validate the approach by studying the in vitro and in vivo phenotypes of EGFR, KRAS and STAG1, genes relevant to cancer biology. This loss-of-function strategy will enable novel experimental strategies in therapeutic target validation and will be instrumental in guiding the lead optimization process by establishing genetic benchmark phenotypes. Citation Format: Ralph Neumüller, Thomas Hoffmann, Alexandra Hörmann, Maja Corcokovic, Johannes Zuber. Precision RNAi using synthetic shRNAmir target sites. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3973.