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RESEARCH PRODUCT

In vitro genome editing using CRISPR/Cas9 to edit SERPINA1 PiZ mutation

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Introduction: The emergence some years ago of the CRISPR/Cas9 system allowed gene therapy to be specific, versatile, cheap and approachable to almost every laboratory. Due to these features, many different genetic diseases such as cystic fibrosis or β-thalassemia have been addressed in cellular models using the CRISPR/Cas9 genetic editing tool. Alpha-1 antytripsin deficiency (AATD) is a rare genetic condition that can provoke respiratory and hepatic symptoms. The Z allele of SERPINA1 gene is a well-characterised point mutation that can trigger the whole pathology. Henceforth, Z mutation is a suitable target for genetic edition using CRISPR/Cas9 in order to develop a gene therapy to treat AATD. Aims and Objectives: The aim of this work is to set-up a CRISPR/Cas9 system to revert the Z mutation on in vitro models. Methods: Five different RNA guides were designed. Hek293 cells were transfected with the RNA guides and GFP using Lipofectamine 3000. After a 48 hour-incubation, transfection efficiency was estimated by fluorescence microscopy. Genomic DNA was isolated and the Z mutation locus was amplified by PCR. Gene editing efficiency was verified by the T7 endonuclease assay. Results: Transfection efficiency was estimated to be approximately 10-20%. After performing T7 endonuclease assay, the digested samples showed two smaller DNA fragments in the agarose gel, indicating that CRISPR/Cas9 system has produced a break in the genome at the Z mutation. Conclusion: SERPINA1 Z mutation can be targeted and edited by the CRISPR/Cas9 system in Hek293 cells with the RNA guides designed. This is the first step towards Z mutation correction in others AATD cellular models. Funding: SVN 2016 and 112/2016 SEPAR grants.