Protocols to evaluate mutant specificity of an oncogene-targeting siRNA using orthogonal in vitro and in vivo approaches

Lyla J Stanland; Alessandro Porrello; Martin Egli; Chad V Pecot

doi:10.1016/j.xpro.2025.104323

Protocols to evaluate mutant specificity of an oncogene-targeting siRNA using orthogonal in vitro and in vivo approaches

STAR Protoc. 2026 Jan 8;7(1):104323. doi: 10.1016/j.xpro.2025.104323. Online ahead of print.

Authors

Lyla J Stanland¹, Alessandro Porrello², Martin Egli³, Chad V Pecot⁴

Affiliations

¹ EnFuego Therapeutics Inc., Morrisville, NC 27560, USA.
² UNC RNA Discovery Center, Chapel Hill, NC 27599, USA; Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
³ Department of Biochemistry, Vanderbilt University, Nashville, TN 37232, USA.
⁴ UNC RNA Discovery Center, Chapel Hill, NC 27599, USA; Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; UNC Division of Oncology, Chapel Hill, NC 27599, USA. Electronic address: pecot@email.unc.edu.

PMID: 41511879
DOI: 10.1016/j.xpro.2025.104323

Abstract

RNA interference (RNAi) is a promising new approach for oncogene targeting for "undruggable" targets, including KRAS. Here, we present a protocol for evaluating mutant selectivity of KRAS small interfering RNAs (siRNAs) using orthogonal in vitro and in vivo techniques. We describe steps for structural analyses of siRNA complexes, utilization of isogenic HA- and luciferase-tagged cell lines, RNA sequencing for off-target effects, and in vivo evaluation of mutant selectivity. This protocol has potential application for the development of mutant-specific siRNA molecules targeting any oncogene. For complete details on the use and execution of this protocol, please refer to Stanland et al.¹.

Keywords: CRISPR; RNA-seq; biotechnology and bioengineering; cancer; cell Biology; cell-based assays; sequence analysis.