Linear donor constructs for targeted integration
Disclosed herein are linear donor molecules comprising homology arms of 50-750 base pairs (e.g., 50-100 base pairs) flanking one or more sequences of interest. The donor molecules and/or compositions comprising these molecules can be used in methods for targeted integration of an exogenous sequence into a specified region of interest in the genome of a cell.
1. A method for homology-dependent targeted integration of a sequence of interest into a region of interest in the genome of a cell, the method comprising the steps of:
(a) expressing a fusion protein in the cell, the fusion protein comprising a meganuclease or zinc finger protein DNA-binding domain and FokI cleavage domain or a cleavage half-domain, wherein the DNA-binding domain has been engineered to bind to a target site in the region of interest;
(b) contacting the cell with a donor polynucleotide comprising a linear donor nucleic acid molecule comprising homology arms of no more than 50 and 100 base pairs in length and a sequence of interest, wherein the homology arms flank the sequence of interest,
wherein binding of the fusion protein to the target site cleaves the genome of the cell in the region of the interest, thereby resulting in homology-dependent targeted integration of the sequence of interest into the genome of the cell.
2. A method for homology-dependent targeted integration of a sequence of interest into a cell, the method comprising:
(a) expressing a first fusion protein in the cell, the first fusion protein comprising a first meganuclease or zinc finger DNA-binding domain and a first FokI cleavage half-domain, wherein the first DNA-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 meganuclease or zinc finger DNA-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
(c) contacting the cell with a polynucleotide comprising a donor nucleic acid comprising a linear donor nucleic acid molecule comprising homology arms of no more than 50 and 100 base pairs in length and a sequence of interest, wherein the homology arms flank the sequence 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 homology-dependent integration of the donor nucleic said into the genome of the cell.
3. The method of claim 1 , wherein at least one DNA-binding domain is a zinc finger binding domain.
4. The method of claim 1 , wherein at least one DNA-binding domain is a meganuclease DNA-binding domain.
5. The method of claim 1 , wherein the sequence of interest from the integrated donor nucleic acid expresses a polypeptide.
6. The method of claim 1 , wherein the sequence in interest from the integrated donor comprises a non-coding nucleic acid sequence.
7. The method of claim 1 , further comprising a cleavage domain from a meganuclease.
8. The method according to claim 1 , wherein the cell is arrested in the G2 phase of the cell cycle.
9. The method according to claim 1 , wherein at least one of the fusion proteins comprises an alteration in the amino acid sequence of the dimerization interface of the cleavage half-domain.
10. The method according to claim 1 , wherein the cell is a mammalian cell.
11. The method according to claim 1 , wherein the cell is a human cell.
12. The method according to claim 1 , wherein the cell is a plant cell.