Genome editing strategies to restore altered splicing events

Cystic fibrosis is an autosomal recessive disease caused by mutations in the CFTR gene. A significant number of mutations (~13%) alter the correct splicing of the CFTR gene, causing the transcription of aberrant transcripts resulting in the production of a non-functional CFTR channel. We focus our research on two intronic CF causing mutations, 3272-26A>G and 3849+10kbC>T that create a new acceptor and donor splice site, respectively, generating in the inclusion of intronic portions into the mRNA. We developed a new genome editing approach to permanently correct the abovementioned mutations by means of CRISPR nucleases. We exploited the use of either Streptococcus pyogenes Cas9, SpCas9, or Acidaminococcus sp. BV3L6, AsCas12a, to edit the aberrant splicing sites and restore the production of the correct transcript, avoiding modifications of the CFTR coding sequence. A comparative analysis between SpCas9 and AsCas12a revealed that the use of AsCas12a with a single crRNA efficiently edits the target loci, producing correctly spliced mRNAs in both 3272-26A>G and 3849+10kbC>T mutations. Furthermore, this genetic repair strategy proved to be highly specific, exhibiting a strong discrimination between the mutated and the wild-type allele and no detectable off-target activity with genome-wide analysis. The selected crRNAs were tested in patients derived primary airway cells and intestinal organoids compound heterozygous for the 3272-26A>G or 3849+10kbC>T mutations, that are considered relevant CF models for translational research. The efficient splicing repair and the complete recovery of CFTR channel activity observed confirmed the goodness of the proposed gene editing strategy. These results demonstrated that allele-specific genome editing with AsCas12a can correct aberrant CFTR splicing mutations, paving the way for a permanent splicing correction in genetic diseases.status: publishe