Fusion proteins and methods for site-directed genome editing
In some aspects, the present invention provides methods and compositions for modifying target sites within nucleic acid molecules. In some embodiments, the methods comprise using adenosine deaminases that act on RNA (ADARs), and variants thereof, to modify target sites within DNA-RNA hybrid molecules. In other aspects, ADAR2 variant polypeptides as well as fusion proteins comprising an ADAR catalytic domain and a hybrid nucleic acid binding domain are provided, as are methods for use thereof. Methods for preventing and treating genetic disorders are also provided herein.
1. A fusion protein comprising a human adenosine deaminase that acts on RNA (ADAR1) catalytic domain linked via an amino acid linker to a hybrid nucleic acid binding domain (NBD) from a human ribonuclease H that binds to a DNA-RNA hybrid molecule.
2. The fusion protein of claim 1 , wherein the human ADAR1 catalytic domain comprises an E1008 mutation and the E1008 position is determined with reference to the sequence set forth in SEQ ID NO:48.
3. A pharmaceutical composition comprising the fusion protein of claim 1 and a pharmaceutically acceptable carrier.
4. A method for preventing or treating a genetic disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 3 .
5. The fusion protein of claim 1 , wherein the human ADAR1 catalytic domain comprises amino acids 833-1226 of SEQ ID NO:48.
6. The fusion protein of claim 1 , wherein the fusion protein modifies a target site without having to introduce a break into the DNA strand of the DNA-RNA hybrid molecule.
7. The fusion protein of claim 1 , wherein the RNA strand of the DNA-RNA hybrid molecule comprises an abasic site.
8. The fusion protein of claim 7 , wherein the human ADAR1 catalytic domain comprises an E1008W, E1008Y, or E1008F mutation and the E1008 position is determined with reference to the sequence set forth in SEQ ID NO:48.
9. The fusion protein of claim 2 , wherein the E1008 mutation comprises an E1008Q mutation.
10. The fusion protein of claim 2 , wherein the E1008 mutation comprises an E1008H mutation.
11. The fusion protein of claim 2 , wherein the E1008 mutation comprises an E1008W, E1008Y, or E1008F mutation.
12. The fusion protein of claim 1 , wherein the human ribonuclease H comprises human ribonuclease H1.