%0 Journal Article
%T ptARgenOM-A Flexible Vector For CRISPR/CAS9 Nonviral Delivery.
%A Radoua A
%A Pernon B
%A Pernet N
%A Jean C
%A Elmallah M
%A Guerrache A
%A Constantinescu AA
%A Hadj Hamou S
%A Devy J
%A Micheau O
%J Small Methods
%V 7
%N 7
%D 2023 07 8
%M 37156748
%F 15.367
%R 10.1002/smtd.202300069
%X 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.