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Generation of a dual-fluorescent reporter cell line to test different delivery approaches for the CRISPR/Cas system

A Alonso-Yanez(1) P Puig-Serra(1) M Martínez-Lage(1) M C Casado-Rosas(1) A Nieto-Sánchez(1) P Ojeda-Walczuk(1) R Torres-Ruiz(1,2) S Rodríguez-Perales(1)

1:Centro Nacional de Investigaciones Oncológicas; 2:CIEMAT-CIBERER

One of the most widely used gene-editing tools is the CRISPR/Cas system, which emerged as an immune prokaryotic defense system against bacteriophages. It is a highly versatile system suitable for multiple applications, including different diagnostic strategies and targeted therapies. However, one of its major concerns is the delivery of the ribonucleoprotein elements when applied for in vivo experiments and gene therapy strategies. In fact, the core of the CRISPR/Cas research is focused on developing appropriate delivery methods and creating reliable models that allow to determine the efficiency of the system when applying these delivery strategies. For this reason, we have generated a dual-fluorescent reporter cell line that has been created through a CRISPR/Cas9-loxP system, with the aim of assessing the efficiency of multiple methods to deliver the CRISPR/Cas elements, such as transient transfection or stable lentivirus infection. Confocal microscopy and flow cytometry analysis were employed to confirm the editing activity through GFP activation when the protein Cas9 was differently delivered. Besides, our preliminary results reveal that the targeted gene-editing events were successfully detected in our in vitro model. In conclusion, this reporter cell line may help to further study and analyse the efficiency of the CRISPR/Cas system in delivery experiments for complex in vivo models, which would increase its translational potential when applied for gene therapy strategies.

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