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.

Abstract Short-hairpin RNA (shRNA) technology enables stable and regulated gene repression through RNA interference (RNAi). Synthetic shRNAs can be embedded into the context of an endogenous microRNA (miRNA) to create natural substrates of RNAi pathways and minimize cellular toxicities. While such “shRNAmir” reagents provide an experimentally versatile RNAi system, their design remains challenging due to our limited understanding of miRNA processing factors. Consequently, shRNAmir reagents often fail to trigger potent target knockdown, especially when expressed from a single genomic integration (“single-copy”), a common scenario in pool-based screens and other key applications. To tackle this issue, we recently developed a high-throughput Sensor assay that enables functional identification of effective shRNAs and has been used to establish shRNA-specific prediction rules. Following these advances in design of the synthetic shRNA stem, here we take a systematic approach to improve the experimental miR-30 backbone. Among several sequence features enhancing knockdown efficiency, we identify a conserved element 3′ of the basal stem that is critically required for optimal shRNAmir processing. Implementation of this feature in an optimized backbone termed “miR-E” results in a 10-30 fold increase in mature shRNA levels, and generally boosts knockdown efficacy. Importantly, combination of the enhanced miR-E backbone with Sensor-based design rules establishes a validated and easily accessible platform for generating single-copy effective shRNAs for pool-based RNAi screening and transgenic animals, and will facilitate the functional annotation of the genome. Citation Format: Christof Fellmann, Thomas Hoffmann, Vaishali Sridhar, Barbara Hopfgartner, Dan Yu Lai, Johannes Zuber. An enhanced microRNA backbone for potent single-copy RNAi. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4273. doi:10.1158/1538-7445.AM2014-4273