Hyperactive piggybac transposases
The present invention provides PiggyBac transposase proteins, nucleic acids encoding the same, compositions comprising the same, kits comprising the same, non-human transgenic animals comprising the same, and methods of using the same.
1. A hyperactive transposase protein comprising at least 90% sequence identity to SEQ ID NO: 2, and comprising an amino acid substitution at position 226 , wherein the substitution at position 226 is a Phenylalanine for a Methionine (M226F), and wherein the hyperactive transposase protein is capable of mediating transposon integration at a higher frequency when compared to a wild type transposase having the sequence of SEQ ID NO: 2.
2. A method of stably integrating an exogenous nucleic acid into the genome of a cell, comprising:
(a) introducing into the cell a transposon comprising the exogenous nucleic acid flanked by a first inverted repeat sequence and a second inverted repeat sequence, wherein the first and second inverted repeat sequences are from piggyBac-like transposons; and
(b) introducing into the cell the hyperactive transposase protein of claim 1 to excise the exogenous nucleic acid and stably integrate the exogenous nucleic acid into the genome.
3. The method of claim 2 , wherein the hyperactive transposase protein is encoded by a nucleic acid that is introduced into the cell.
4. The method of claim 3 , wherein the transposon is present on a vector and the hyperactive transposase protein is introduced as a transposase RNA.
5. The method of claim 2 , wherein the transposon is present on a vector, and the hyperactive transposase protein is introduced as an expressed protein.
6. The hyperactive transposase protein of claim 1 , wherein the hyperactive transposase protein further comprises an amino acid substitution at positions 30 , 165 and 282 , wherein the substitution at position 30 is a substitution of a Valine for an Isoleucine (I30V), wherein the substitution at position 165 is a substitution of a Serine for a Glycine (G165S), wherein the substitution at position 282 is a substitution of any amino acid (X) for a Methionine (M282X), the amino acid (X) selected from the group consisting of a Leucine (L), an Isoleucine (I), a Valine (V), and an Alanine (A).
7. A nucleic acid encoding the hyperactive transposase protein of claim 1 .
8. A nucleic acid encoding the hyperactive transposase protein of claim 6 .
9. A vector comprising the nucleic acid of claim 7 .
10. A vector comprising the nucleic acid of claim 8 .
11. A cell comprising the hyperactive transposase protein of claim 1 .
12. A cell comprising the hyperactive transposase protein of claim 6 .
13. A cell comprising the nucleic acid of claim 7 .
14. A cell comprising the nucleic acid of claim 8 .
15. The vector of claim 9 , wherein the vector is a plasmid, a liposome, a virus or a nanoparticle.
16. The vector of claim 10 , wherein the vector is a plasmid, a liposome, a virus or a nanoparticle.