Targeted integration and expression on exogenous nucleic acid sequences
Disclosed herein are methods and compositions for targeted integration of a exogenous sequence into a predetermined target site in a genome for use, for example, in protein expression and gene inactivation.
1. An in vitro method for expressing a product of an exogenous nucleic acid sequence in a mammalian cell, the method comprising:
(a) expressing a first fusion protein in the cell, the first fusion protein comprising a first zinc finger binding domain and a first FokI cleavage half-domain, wherein the first zinc finger binding domain has been engineered to bind to a first target site in a region of interest in the genome of the cell, wherein the first cleavage half-domain comprises a mutation in the wild-type amino acid residue at position 486 to a Glu (E) residue and further wherein the region of interest is in an endogenous region of the genome that is transcriptionally active and not essential for viability;
(b) expressing a second fusion protein in the cell, the second fusion protein comprising a second zinc finger binding domain and a second FokI cleavage half domain, wherein the second zinc finger binding domain binds to a second target site in the region of interest in the genome of the cell, wherein the second target site is different from the first target site, and further wherein the second cleavage domain comprises a mutation in the wild-type amino acid residue at position 490 to a Lys (K) residue; and
(c) contacting the cell with a polynucleotide comprising the exogenous nucleic acid sequence and a first nucleotide sequence that is homologous to a first sequence in the region of interest;
wherein binding of the first fusion protein to the first target site, and binding of the second fusion protein to the second target site, positions the cleavage half-domains such that the genome of the cell is cleaved in the region of interest, thereby resulting in integration of the exogenous sequence into the genome of the cell in the region of interest and expression of the product of the exogenous sequence.
2. The method according to claim 1 , wherein the polynucleotide further comprises a second nucleotide sequence that is homologous to a second sequence in the region of interest.
3. The method according to claim 2 , wherein the first and second nucleotide sequences flank the exogenous sequence.
4. The method according to claim 1 , wherein the region of interest is a CCR5 gene.
5. The method according to claim 1 , wherein the cell is arrested in the G2 phase of the cell cycle.
6. An in vitro method for integrating an exogenous sequence into a region of interest in the genome of a mammalian cell, the method comprising:
(a) expressing a first fusion protein in the cell, the first fusion protein comprising a first zinc finger binding domain and a first cleavage half-domain, wherein the first zinc finger binding domain has been engineered to bind to a first target site in a region of interest in the genome of the cell;
(b) expressing a second fusion protein in the cell, the second fusion protein comprising a second zinc finger binding domain and a second cleavage half domain, wherein the second zinc finger binding domain binds to a second target site in the region of interest in the genome of the cell, wherein the second target site is different from the first target site; and
(c) contacting the cell with a polynucleotide comprising an exogenous nucleic acid sequence, wherein the exogenous nucleic acid sequence comprises a meganuclease recognition site;
wherein the first and second fusion proteins comprise an alteration in the amino acid sequence of the dimerization interface of the cleavage half-domain, wherein the cleavage half-domain of the first fusion protein is a FokI cleavage half-domain and further wherein the alteration comprises mutating the wild-type amino acid residue at position 486 to a Glu (E) residue and wherein the cleavage half-domain of the second fusion protein is a FokI cleavage half-domain and further wherein the alteration comprises mutating the wild-type amino acid residue at position 490 to a Lys (K) residue; and further wherein binding of the first fusion protein to the first target site, and binding of the second fusion protein to the second target site, positions the cleavage half-domains such that the genome of the cell is cleaved in the region of interest, thereby resulting in integration of the exogenous sequence into the genome of the cell in the region of interest.
7. The method according to claim 6 , wherein the integration inactivates gene expression in the region of interest.
8. The method according to claim 6 , wherein the exogenous nucleic acid sequence is between 10 and 50 nucleotides in length.
9. The method according to claim 6 , wherein the meganuclease is I-SceI.
10. The method according to claim 6 , wherein the meganuclease has been engineered to bind a non-natural target site.
11. The method according to claim 6 , wherein the cell is arrested in the G2 phase of the cell cycle.