ptARgenOM—A Flexible Vector For CRISPR/CAS9 Nonviral Delivery - Inserm - Institut national de la santé et de la recherche médicale
Article Dans Une Revue Small Methods Année : 2023

ptARgenOM—A Flexible Vector For CRISPR/CAS9 Nonviral Delivery

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.
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inserm-04138961 , version 1 (23-06-2023)

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Abdelmnim Radoua, Baptiste Pernon, Nicolas Pernet, Chloé Jean, Mohammed Elmallah, et al.. ptARgenOM—A Flexible Vector For CRISPR/CAS9 Nonviral Delivery. Small Methods, 2023, 7 (7), pp.e2300069. ⟨10.1002/smtd.202300069⟩. ⟨inserm-04138961⟩
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