PT  - JOURNAL ARTICLE
AU  - Lucia Banyuls
AU  - Daniel Pellicer
AU  - Silvia Castillo
AU  - María Magallón
AU  - María Mercedes Navarro
AU  - Amparo Escribano
AU  - Francisco Dasí
TI  - In vitro genome editing using CRISPR/Cas9 to edit SERPINA1 PiZ mutation
AID  - 10.1183/13993003.congress-2019.PA5411
DP  - 2019 Sep 28
TA  - European Respiratory Journal
PG  - PA5411
VI  - 54
IP  - suppl 63
4099  - http://erj.ersjournals.com/content/54/suppl_63/PA5411.short
4100  - http://erj.ersjournals.com/content/54/suppl_63/PA5411.full
SO  - Eur Respir J2019 Sep 28; 54
AB  - 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.FootnotesCite this article as: European Respiratory Journal 2019; 54: Suppl. 63, PA5411.This is an ERS International Congress abstract. No full-text version is available. Further material to accompany this abstract may be available at www.ers-education.org (ERS member access only).