ptARgenOM—A Flexible Vector For CRISPR/CAS9 Nonviral Delivery

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. Nonviral CRISPR‐Cas9 gene editing of intracellular proteins often relies on the amplification of single‐cell clones, which can be time‐consuming and laborious. ptARgenOM, is a flexible all‐in‐one delivery vector that allows transient selection and enrichment of knockout cells using either enhanced green fluorescent protein or puromycin. This bimodal delivery vector provides a simple, fast, and cost‐effective delivery tool for genome editing.