Site-directed CRISPR/recombinase compositions and methods of integrating transgenes
Disclosed herein are targeted chimeric polypeptides, compositions thereof, expression vectors, and methods of use thereof, for the generation of transgenic cells, tissues, plants, and animals. The compositions, vectors, and methods of the present invention are also useful in gene therapy and cell therapy techniques. The chimeric polypeptide includes a CRISPR-Cas domain and a recombinase domain.
1. A polypeptide comprising:
(i) a catalytically dead CRISPR-Cas domain having an amino acid sequence of SEQ ID NO. 1 with a D1OA mutation and a second mutation selected from H840A, N854A, N863A; and
(ii) a recombinase domain comprising a serine recombinase, wherein the serine recombinase is Tn3 (SEQ ID NO: 3) and wherein the recombinase domain has an amino acid sequence selected from the group consisting of SEQ ID NO: 5 and the following variants thereof:
(a) I 77 L
(b) F83L F107L
(c) I3T D95E
(d) G7OS F107L
(e) E57G A89V
(f) K37R D44N
(g) I97V I103T F107L
(h) G70S
(i) F107L
(j) E57G
(k) A89V
(l) I77L I3P V108A
(m) I77L L135R
(n) I77L S12R 1103V
(o) I77L E132A
(p) I77L I3L L135R
(q) I77L I3S E132A; and
(r) I77L N127H E132A.
2. The polypeptide of claim 1 , further comprising a linker that links the CRISPR-Cas domain and the recombinase domain.
3. The polypeptide of claim 2 , wherein the linker has an amino acid sequence of The-Ser.
4. A composition comprising: a first nucleotide sequence encoding the polypeptide of claim 1 .
5. The composition of claim 4 , further comprising a donor DNA comprising a transgene.
6. A method of integrating a transgene into the genome of a cell comprising introducing into the cell the composition of claim 4 .
7. The method of claim 6 , wherein the cell is a eukaryotic cell.
8. The method of claim 6 , wherein the cell is a mammalian or human cell.
9. The method of claim 6 , wherein the cell is a one-cell embryo.