Modified nucleic acid editing systems for tethering donor DNA
The technology relates to a composition for tethering donor DNA to a nuclease, the composition comprising a nucleic acid comprising donor DNA and a consensus sequence for a DNA binding domain; and at least one of: a fusion protein comprising a nuclease coupled to a DNA binding domain for binding the consensus sequence; and a nucleic acid encoding the fusion protein.
1. A composition for tethering donor DNA to a nuclease, the composition comprising a nucleic acid comprising donor DNA and a consensus sequence for a DNA binding domain; and at least one of:
a fusion protein a fusion protein comprising a nuclease and further comprising a DNA binding domain for binding the consensus sequence, wherein the DNA binding domain consists of amino acids 43-403 of SEQ ID NO: 9; and
a nucleic acid encoding the fusion protein.
2. The composition of claim 1 wherein the nuclease is a Cas protein, a Transcription activator-like effector nuclease (TALEN), a meganuclease, or a Zinc Finger.
3. The composition of claim 1 wherein the nuclease is a Cas protein.
4. The composition of claim 3 wherein the Cas protein is Cas9.
5. The composition of claim 1 wherein the fusion protein further comprises a nuclear localization sequence.
6. The composition of claim 2 , further comprising a guide RNA that interacts with the Cas protein and a target DNA sequence.
7. The composition of claim 1 wherein the consensus sequence comprises a Lac operator.
8. The composition of claim 7 wherein the consensus sequence comprises a sequence with at least 80%, 85%, 90%, 95% or at least 99% identity to the Lac operator as set forth in SEQ ID NO: 66.
9. The composition of claim 1 wherein the nuclease is coupled to the DNA binding domain via a linker.
10. The composition of claim 9 wherein the linker comprises a sequence selected from any one of SEQ ID NOs: 3 to 7, a GGS linker, or amino acids 404-419 of SEQ ID NO: 9.
11. The composition of claim 1 wherein the fusion protein comprises a LAC repressor and a Cas9.
12. The composition of claim 1 comprising a vector, wherein either or both of:
a. the nucleic acid comprising the donor DNA and the consensus sequence for a DNA binding domain; and
b. the nucleic acid encoding the fusion protein, are present in a vector.
13. The composition of claim 12 wherein the vector further comprises a nucleic acid sequence encoding a guide RNA that interacts with the Cas protein and a target DNA sequence.
14. The composition of claim 1 wherein the nuclease is modified to reduce or eliminate nuclease activity.
15. A kit comprising:
a nucleic acid comprising donor DNA and a consensus sequence for a DNA binding domain; and at least one of
a fusion protein a fusion protein comprising a nuclease and further comprising a DNA binding domain for binding the consensus sequence, wherein the DNA binding domain consists of amino acids 43-403 of SEQ ID NO: 9; and
a nucleic acid encoding the fusion protein.
16. The kit of claim 15 wherein the nuclease is a Cas protein, a Transcription activator-like effector nuclease (TALEN), a meganuclease, or a Zinc Finger.
17. The kit of claim 16 wherein the Cas protein is Cas9.