Work

An optimized pipeline for cell-based validation of gRNAs designs to inactivate coding and non-coding RNAs using CRISPR-Cas9

Year: 2026

Type: article

Abstract: Genome-editing technologies based on bacterial Cas9 proteins have become a gold standard for introducing precise genetic modifications across multiple species and experimental models. These tools require the design and validation of guide RNAs (gRNAs) that direct the Cas9 protein to the actual genome location to be edited. Although numerous algorithms have been developed to design gRNAs, whether a gRNA is going to be effective or not need to be experimentally assessed. Since most of the times several designs are tested in parallel, we have set up an efficient cell-based approach to optimize this validation process by directly quantifying gRNAs effect on the downstream target RNA or protein. Our strategy relies on: (1) creating stable cell lines expressing the gene of interest and a fluorescent reporter; (2) transient transfection of these lines with the different gRNAs; (3) analyzing the efficiency of the designs at the transcriptomic and/or protein level in the whole population or in FACS sorted cells. We demonstrate the relevance of our approach by inactivating a coding gene (Elk1) and two short non-coding RNA (miR-218 and miR-124).

Source: Scientific Reports

DOI: 10.1038/s41598-026-73063-z