Influencing factors for CRISPR/Cas9 efficacy for F508del mutation editing in cystic fibrosis

Symptomatic and pathogenetic treatment of cystic fibrosis allowed to increase patients’ lifespan up to 30 years, but disease is still incurable. New gene therapy technologies based on the use of specific nucleases open up new opportunities in development of etiology-based therapy for hereditary diseases. Among these methods CRISPR/Cas9 is the most widely used approach for genome editing. The aim of the study is to compare correction efficacy of CFTR gene by Cas9 with different guide RNAs (sgRNAs), designed to F508del mutation, and to increase their activity. We used modified spCas9 (eSpCas9) and two sgRNAs designed to CFTR gene: sgCFTR#1 - directly to the F508del mutation and sgCFTR#2 - 14 nucleotides 5’-upstream from the mutation. SgGFP designed to GFP gene was used as a control of the nuclease activity. PGEM-TA-CFTR plasmid with part of the CFTR gene with the F508del mutation co-transfected with the plasmid for CRISPR/Cas9 was used as template for sgCFTR#1 and #2; plasmid pEGFP-C1 was used as template for sgGFP. Experiments performed in HEK293T cell culture demonstrated, that sgCFTR#1 has the lowest efficiency among used sgRNAs - number of insertions/deletions (indels) by T7E1 assay was 6.37-20.82%. Expression level of sgCFTR#1 after transfection was lower than expression of sgGFP, which showed the greatest activity - up to 65% of indels. Addition of G-quadruplexes to sgCFTR#1 and sgGFP sequences with the aim to increase their stability led to decrease of expression and activity. Culturing transfected cells at lower temperature (24 hours at 37°C, then 48 hours at 30°C) resulted in two-fold decrease of sgCFTR#1 activity, but without affecting sgGFP activity. Thus, direct relationship between sgRNAs expression and their activity was confirmed in the study; however, sgCFTR#1 expression and its efficacy could not be increased. Further attempts to enhance sgCFTR#1 expression and its stabilization should be performed, or other Cas9 enzymes, which expand the ability to select sgRNA direct to F508del mutation can be potentially used.

муковисцидоз, CRISPR/Cas9, экспрессия sgRNA, CFTR, G-квадруплекс, сystic fibrosis, CRISPR/Cas9, sgRNA expression, CFTR, G-quadruplex

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