METHODS FOR CORRECTING VON WILLEBRAND FACTOR POINT MUTATIONS
First Claim
1. A method of editing a nucleic acid molecule encoding a von Willebrand Factor (vWF) protein, the method comprising contacting the nucleic acid molecule with(a) a fusion protein comprising a nuclease-inactive Cas9 domain and a deaminase domain;
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wherein the nucleic acid molecule comprises a T>
C and/or an A>
G point mutation in the vWF-encoding nucleic acid molecule as compared to a wild-type vWF-encoding nucleic acid molecule,and wherein the vWF-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.
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Abstract
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 von Willebrand Factor protein to correct a point mutation associated with a disease or disorder, e.g., with von Willebrand disease. The methods provided are useful for correcting a vWF 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.
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Citations
22 Claims
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1. A method of editing a nucleic acid molecule encoding a von Willebrand Factor (vWF) 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 vWF-encoding nucleic acid molecule; wherein the nucleic acid molecule comprises a T>
C and/or an A>
G point mutation in the vWF-encoding nucleic acid molecule as compared to a wild-type vWF-encoding nucleic acid molecule,and wherein the vWF-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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)
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Specification