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Modeling of cystic fibrosis in HEK293t cell culture and of a method for the correction of F508del mutation

[Cystic fibrosis]
Svetlana Anatolevna Smirnikhina; Ekaterina Vladimirovna Kondratyeva; Arina Arturovna Anuchina; Milyausha Irshatovna Zaynitdinova; Alexander Vyacheslavovich Lavrov;

The work was carried out to study the possibility of modeling of cystic fibrosis in HEK293T cell culture and the correction of the F508del mutation using the CRISPR/Cas9 method. Four sgRNAs in combination with three Cas9 nucleases and two DNA repair templates were tested. Modeling of cystic fibrosis in HEK293T cell culture was achieved by transfection of an additional plasmid pGEM-CFTR containing the CFTR locus with the F508del mutation into cells. The efficacy of mutation editing was assessed using deep targeted sequencing. The average editing efficiency of the CFTR locus was 5.5 %. The mutation correction frequency ranged from 0.08 % to 0.7 % alleles, depending on the combination of used CRISPR/Cas9 components. The accuracy of different Cas9/sgRNA combinations ranged from 61.4 % to 90.9 %, the maximum value was recorded for saCas9/saCFTR#3. The possibility of modeling cystic fibrosis in a HEK293T cell culture using a synthetic plasmid was demonstrated and the correction efficiency of the F508del mutation using various CRISPR/Cas9 combinations was evaluated

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Keywords: fibrosis, CFTR, genome editing, CRISPR/Cas9, F508del, HEK293T


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Pyatigorsk State Research Institute of Balneotherapeutics
Pyatigorsk State Pharmaceutical Academy