Cas9 fusion molecules, gene editing systems, and methods of use thereof

1. An enzymatically active Cas9 (eaCas9) fusion molecule comprising an eaCas9 molecule covalently linked to a template nucleic acid by a polypeptide linker.

2. The eaCas9 fusion molecule of claim 1 , wherein

i) the eaCas9 molecule comprises at least one surface exposed cysteine residue;

ii) the eaCas9 molecule comprises succinimidyl-6-hydrazino-nicotinamide (S-HyNic); or

iii) the eaCas9 molecule comprises a tag linked to the eaCas9 molecule.

3. The eaCas9 fusion molecule of claim 1 wherein the template nucleic acid comprises a double stranded nucleic acid or a single stranded nucleic acid.

4. The eaCas9 fusion molecule of claim 1 , wherein the eaCas9 molecule is a wild-type Cas9 molecule, a Cas9 nickase molecule, a split Cas9 molecule, or an inducible Cas9 molecule.

5. A gene editing system, comprising

at least one enzymatically active Cas9 (eaCas9) fusion molecule comprising an eaCas9 molecule covalently linked to a template nucleic acid by a polypeptide linker, and

at least one gRNA molecule.

6. The gene editing system of claim 5 ,

i) wherein the at least one gRNA molecule and the eaCas9 fusion molecule are designed to associate with a target nucleic acid and generate a double strand break on the target nucleic acid, wherein the double strand break is repaired by at least one DNA repair pathway, thereby producing a modified target nucleic acid; or

ii) wherein the eaCas9 molecule is a Cas9 nickase molecule.

7. A pharmaceutical composition comprising the gene editing system of claim 5 .

8. The eaCas9 fusion molecule of claim 1 , wherein

i) the polypeptide linker is between 3 and 100 amino acids in length; or

ii) the polypeptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 206-214.

9. The eaCas9 fusion molecule of claim 2 , wherein the eaCas9 molecule comprises a tag linked to the eaCas9 molecule; and

the template nucleic acid comprises a haloalkane; and

the tag comprises a HaloTag molecule,

wherein the HaloTag molecule is linked to the haloalkane-containing template nucleic acid using an SN 2 reaction.

10. A gene editing system, comprising

(i) a first eaCas9 fusion molecule, wherein the first eaCas9 fusion molecule comprises a first Cas9 nickase molecule covalently linked to a template nucleic acid by a polypeptide linker;

a first gRNA molecule;

a second eaCas9 fusion molecule, wherein the second eaCas9 fusion molecule comprises a second Cas9 nickase molecule covalently linked to the template nucleic acid by a polypeptide linker; and

a second gRNA molecule; or

(ii) a first eaCas9 fusion molecule, wherein the first eaCas9 fusion molecule comprises a first Cas9 nickase molecule covalently linked to a first template nucleic acid by a polypeptide linker;

a first gRNA molecule;

a second eaCas9 fusion molecule, wherein the second eaCas9 fusion molecule comprises a second Cas9 nickase molecule covalently linked to a second template nucleic acid by a polypeptide linker; and

a second gRNA molecule.

11. A method of modifying a target nucleic acid in a cell, the method comprising:

contacting the cell with a gRNA molecule and the eaCas9 fusion molecule of claim 1 ;

wherein the gRNA molecule and the eaCas9 fusion molecule associate with the target nucleic acid and generate a double strand break in the target nucleic acid; and

wherein the double strand break is repaired by gene correction using the template nucleic acid of the eaCas9 fusion molecule.

12. A method of modifying a target nucleic acid in a cell, the method comprising:

contacting the cell with a first gRNA molecule; a first eaCas9 molecule; a second gRNA molecule; and a second eaCas9 molecule;

wherein at least one of the first and second eaCas9 molecule is covalently linked to a template nucleic acid by a polypeptide linker,

wherein the first gRNA molecule and the first eaCas9 molecule associate with the target nucleic acid and generate a first single strand cleavage event on a first strand of the target nucleic acid;

wherein the second gRNA molecule and the second eaCas9 molecule associate with the target nucleic acid and generate a second single strand cleavage event on a second strand of the target nucleic acid, thereby forming a double strand break having a first overhang and a second overhang; and wherein the first overhang and the second overhang in the target nucleic acid are repaired by gene correction using the template nucleic acid.

13. The method of claim 12 , wherein the first eaCas9 molecule is covalently linked to the template nucleic acid by a polypeptide linker.

14. The method of claim 12 , wherein both the first eaCas9 molecule and the second eaCas9 molecule are covalently linked to the template nucleic acid by a polypeptide linker.