METHODS FOR CORRECTING CASPASE-9 POINT MUTATIONS
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 Caspase-9 protein to correct a point mutation associated with a disease or disorder, e.g., with neuroblastoma. The methods provided are useful for correcting a Caspase-9 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.
1 . A method of editing a nucleic acid molecule encoding a Caspase-9 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 Caspase-9-encoding nucleic acid molecule;
wherein the nucleic acid molecule comprises a T>C and/or an A>G point mutation in the Caspase-9-encoding nucleic acid molecule as compared to a wild-type Caspase-9-encoding nucleic acid molecule,
and wherein the Caspase-9-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.
2 . The method of claim 1 , wherein the deaminase is a cytidine deaminase.
3 . The method of claim 1 , wherein the deaminase is an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase.
4 . The method of claim 3 , wherein the deaminase is an APOBEC1 family deaminase.
5 . The method of claim 3 , wherein the deaminase is an activation-induced cytidine deaminase (AID).
6 . The method of claim 1 , wherein the deaminase is an ACF1/ASE deaminase.
7 . The method of claim 1 , wherein the deaminase is an adenosine deaminase.
8 . The method of claim 7 , wherein the deaminase is an ADAT family deaminase.
9 . The method of claim 1 , wherein the deaminase domain is fused to the N-terminus of the Cas9 domain.
10 . The method of claim 1 , wherein the deaminase domain is fused to the C-terminus of the Cas9 domain.
11 . The method of claim 1 , wherein the Cas9 domain and the deaminase domain are fused via a linker.
12 . The method of claim 1 , wherein the linker comprises a (GGGGS)n (SEQ ID NO: 91), a (G)n, an (EAAAK)n (SEQ ID NO: 5), or an (XP)n motif, or a combination of any of these, wherein n is independently an integer between 1 and 30.
13 . The method of claim 1 , wherein the T>C and/or an A>G point mutation in the Caspase-9-encoding nucleic acid molecule is associated with a disease or disorder, and wherein the deamination of the mutant C or G residue results in a sequence that is not associated with a disease or disorder.
14 . The method of claim 1 , wherein the deamination results in a correction of the T>C and/or an A>G point mutation to restore the wild-type sequence.
15 . The method of claim 1 , wherein the sequence associated with the disease or disorder encodes a protein, and wherein the deamination introduces a stop codon into the sequence associated with the disease or disorder, resulting in a truncation of the encoded protein.
16 . The method of claim 1 , wherein the T>C and/or an A>G point mutation causes an amino acid sequence in the Caspase-9 protein as compared to the wild type Caspase-9 protein.
17 . The method of claim 1 , wherein the Caspase-9 protein comprises an L197P substitution caused by an T>C point mutation in codon 197 of the Caspase-9 gene.
18 . The method of claim 17 , wherein the Caspase-9 point mutation is associated with neuroblastoma.
19 . The method of claim 17 , wherein the contacting results in deamination of the mutant cytidine residue in codon 197 of the Caspase-9 gene, thus correcting the T>C point mutation.
20 . The method of claim 1 , wherein the contacting is in vivo in a subject having or diagnosed with the disease or disorder.
21 . The method of claim 1 , wherein the method further comprises detecting the deamination the mutant C or G nucleotide base.
22 . The method of claim 21 , wherein the detecting is via PCR.