Fad2 performance loci and corresponding target site specific binding proteins capable of inducing targeted breaks
Methods and compositions for gene disruption, gene editing or gene stacking within a FAD2 loci by cleaving, in a site directed manner, a location in a FAD2 gene in a soybean cell, to generate a break in the FAD2 gene and then optionally integrating into the break a nucleic acid molecule of interest is disclosed.
1. A zinc finger protein that binds to a nucleic acid target site comprising any of SEQ ID NO: 14 to SEQ ID NO: 20.
2. The zinc finger protein of claim 1 , wherein the zinc finger protein comprises from three to six zinc finger domains, each zinc finger domain comprising a recognition helix region, wherein the zinc finger protein comprises the recognition helix regions ordered and shown in a single row of Table 2.
3. A zinc finger nuclease comprising a zinc finger protein of claim 1 and a cleavage domain.
4. A polynucleotide comprising a sequence encoding a zinc finger protein according to claim 1 .
5. A polynucleotide comprising a sequence encoding a zinc finger nuclease according to claim 3 .
6. An isolated cell comprising a zinc finger protein according to claim 1 .
7. An isolated cell comprising a polynucleotide according to claim 4 .
8. A method of cleaving a FAD2 loci in a soybean cell, the method comprising:
introducing a polynucleotide encoding the zinc finger nuclease (ZFN) according to claim 3 into the cell, wherein the ZFN cleaves the FAD2 loci and further wherein the ZFN comprises a pair of zinc finger proteins as shown in Table 2 as follows: a pair comprising the ZFPs designated 37354 and 37355; a pair comprising the ZFPs designated 37370 and 37371; a pair comprising the ZFPs designated 37374 and 37375; a pair comprising the ZFPs designated 37366 and 37367; a pair comprising the ZFPs designated 37384 and 37385; a pair comprising the ZFPs designated 37398 and 37399; or a pair comprising the ZFPs designated 37392 and 37393.
9. The method according to claim 8 , further comprising integrating a nucleic acid molecule of interest into the cleaved FAD2 loci.
10. The method according to claim 9 , wherein the FAD2 loci is a FAD2 2.3 gene, FAD2 2.6 gene, or both.
11. The method according to claim 10 , wherein the cleavage domain is selected from the group consisting of a cleavage half-domain from a type IIS restriction endonuclease, a cleavage half-domain from Fold endonuclease, a cleavage half-domain from StsI endonuclease, and a homing endonuclease.