METHODS FOR CORRECTING ALPHA-ANTITRYPSIN POINT MUTATIONS

摘要

Some aspects of this disclosure provide strategies, systems, reagents, methods, and kits that are useful for the targeted editing of nucleic acids, including editing a nucleic acid encoding a mutant α-antitrypsin protein to correct a point mutation associated with a disease or disorder, e.g., with chronic obstructive pulmonary disease (COPD) disease. The methods provided are useful for correcting an α-antitrypsin point mutation within the genome of a cell or subject, e.g., within the human genome. In some embodiments, fusion proteins of Cas9 and nucleic acid editing enzymes or enzyme domains, e.g., deaminase domains, are provided. In some embodiments, reagents and kits for the generation of targeted nucleic acid editing proteins, e.g., fusion proteins of Cas9 and nucleic acid editing enzymes or domains, are provided.

主权项

1. A method of editing a nucleic acid molecule encoding an α-antitrypsin protein, the method comprising contacting the nucleic acid molecule with
(a) a fusion protein comprising a nuclease-inactive Cas9 domain and a deaminase domain; and (b) an sgRNA targeting the fusion protein of (a) to the α-antitrypsin-encoding nucleic acid molecule; wherein the nucleic acid molecule comprises a T>C and/or an A>G point mutation in the α-antitrypsin-encoding nucleic acid molecule as compared to a wild-type α-antitrypsin-encoding nucleic acid molecule, and wherein the α-antitrypsin-encoding nucleic acid molecule is contacted with the fusion protein and the sgRNA in an amount effective and under conditions suitable for the deamination the mutant C or G nucleotide base.