Linear donor constructs for targeted integration

Abstract

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 linear donor nucleic acid molecule comprising homology arms of between 50 and 750 base pairs and a sequence of interest, wherein the homology arms flank the sequence of interest. 
     
     
         2 . The linear donor nucleic acid of  claim 1 , wherein the homology arms are between 50 and 100 base pairs in length. 
     
     
         3 . The linear donor nucleic acid of  claim 1 , wherein one or more of the base pairs of the homology arms are joined with a phosphorothioate phosphodiester bond. 
     
     
         4 . The linear donor nucleic acid of  claim 3 , wherein the phosphorothioate phosphodiester bonds are positioned at the first and, optionally, second bonds of the 5′ and 3′ ends of the donor nucleic acid. 
     
     
         5 . The linear donor nucleic acid of  claim 1 , further comprising, between the homology arms, a sequence encoding a 2A peptide. 
     
     
         6 . The linear donor nucleic acid of  claim 1 , further comprising, between the homology arms, a sequence comprising an SA site. 
     
     
         7 . The linear donor nucleic acid of  claim 1 , further comprising, between the homology arms, a sequence comprising an IRES sequence. 
     
     
         8 . The linear donor nucleic acid of  claim 1 , wherein the sequence of interest does not encode a polypeptide. 
     
     
         9 . The linear donor nucleic acid of  claim 1 , further comprising a promoter sequence operably linked to the sequence of interest. 
     
     
         10 . The linear donor nucleic acid of  claim 1 , wherein the sequence of interest encodes a polypeptide. 
     
     
         11 . The linear donor nucleic acid according to  claim 10 , wherein the polypeptide is selected from the group consisting of an antibody, an antigen, an enzyme, a growth factor, a cell surface receptor, a nuclear receptor, a hormone, a lymphokine, a cytokine, a reporter gene, a selectable marker, a secreted factor, an epitope tag and functional fragments thereof and combinations thereof. 
     
     
         12 . The linear donor nucleic acid of  claim 1 , wherein the sequence contains a non-coding nucleic acid. 
     
     
         13 . The linear donor nucleic acid according to  claim 12 , wherein the non-coding nucleic acid is selected from the group consisting of a miRNA, and SH-RNA, or siRNA. 
     
     
         14 . A method for homology-dependent targeted integration of a sequence of interest into a region of interest in the genome of the cell, the method comprising the steps of:
 (a) expressing a fusion protein in the cell, the fusion protein comprising a DNA-binding domain and 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 of  claim 1 ,   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.   
     
     
         15 . 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 DNA-binding domain and a first 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 DNA-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 a donor nucleic acid according to  claim 1 ;   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.   
     
     
         16 . The method of  claim 14 , wherein at least one DNA-binding domain is a zinc finger binding domain. 
     
     
         17 . The method of  claim 14 , wherein at least one DNA-binding domain is a meganuclease DNA-binding domain. 
     
     
         18 . The method of  claim 14 , wherein the sequence of interest from the integrated donor nucleic acid expresses a polypeptide. 
     
     
         19 . The method of  claim 14 , wherein the sequence in interest from the integrated donor comprises a non-coding nucleic acid sequence. 
     
     
         20 . The method of  claim 14 , wherein the cleavage domain is from a meganuclease. 
     
     
         21 . The method of  claim 14 , wherein the first and second cleavage half-domains are from a Type IIS restriction endonuclease. 
     
     
         22 . The method according to  claim 21 , wherein the Type IIS restriction endonuclease is selected from the group consisting of FokI and StsI. 
     
     
         23 . The method according to  claim 14 , wherein the cell is arrested in the G2 phase of the cell cycle. 
     
     
         24 . The method according to  claim 14 , 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. 
     
     
         25 . The method according to  claim 14 , wherein the cell is a mammalian cell. 
     
     
         26 . The method according to  claim 14 , wherein the cell is a human cell. 
     
     
         27 . The method according to  claim 14 , wherein the cell is a plant cell.