Fiche publication
Date publication
mai 2023
Journal
Small methods
Auteurs
Membres identifiés du Cancéropôle Est :
Dr DEVY Jérôme
,
Dr MICHEAU Olivier
,
Mr PERNET Nicolas
Tous les auteurs :
Radoua A, Pernon B, Pernet N, Jean C, Elmallah M, Guerrache A, Constantinescu AA, Hadj Hamou S, Devy J, Micheau O
Lien Pubmed
Résumé
Viral-mediated delivery of the CRISPR-Cas9 system is one the most commonly used techniques to modify the genome of a cell, with the aim of analyzing the function of the targeted gene product. While these approaches are rather straightforward for membrane-bound proteins, they can be laborious for intracellular proteins, given that selection of full knockout (KO) cells often requires the amplification of single-cell clones. Moreover, viral-mediated delivery systems, besides the Cas9 and gRNA, lead to the integration of unwanted genetic material, such as antibiotic resistance genes, introducing experimental biases. Here, an alternative non-viral delivery approach is presented for CRISPR/Cas9, allowing efficient and flexible selection of KO polyclonal cells. This all-in-one mammalian CRISPR-Cas9 expression vector, ptARgenOM, encodes the gRNA and the Cas9 linked to a ribosomal skipping peptide sequence followed by the enhanced green fluorescent protein and the puromycin N-acetyltransferase, allowing for transient, expression-dependent selection and enrichment of isogenic KO cells. After evaluation using more than 12 distinct targets in 6 cell lines, ptARgenOM is found to be efficient in producing KO cells, reducing the time required to obtain a polyclonal isogenic cell line by 4-6 folds. Altogether ptARgenOM provides a simple, fast, and cost-effective delivery tool for genome editing.
Mots clés
CRISPR/Cas9, cell sorting, gene editing, guide RNA, intracellular gene products, polyclonal cells, puromycin selection
Référence
Small Methods. 2023 05 8;:e2300069
