CRISPR-Cas9 nuclease offers the opportunity to edit the genomic DNA with high efficiency and precision. The most common mutation causing Cystic Fibrosis is the deletion of a phenylalanine in position 508 of the CFTR protein (F508del) and the current available technologies are not efficient for the correction of this type of mutations (3 nucleotides deletion). Here, researchers propose to restore the activity of the F508del CFTR by the development of a new strategy which rely on the existence of neutralizing mutations. Indeed, previous reports showed that folding and trafficking defects caused by F508del CFTR can be partially corrected by secondary point mutations in the CFTR gene (neutralizing mutations). In order to identify new mutations able to restore F508del CFTR channel activity, researchers will exploit a molecular circuit, called EvolvR and based on CRISPR-Cas9, to insert new random mutations into the CFTR sequence and to specifically select those able to promote the correct trafficking of the protein to the plasma membrane. A functional screening will be performed to isolate the mutations that best restore the activity of CFTR channel. Selected mutations will be inserted into cellular models for cystic fibrosis carrying the F508del mutation (primary airway epithelial cells and patients’ intestinal organoids) by mean of CRISPR-base editor, that allow targeted single-base DNA modifications.