Methods and compositions for targeted polynucleotide modification

Patent metadata

Field	Value
Publication number	US-10443064-B2
Application number	US-201815890698-A
Country	US
Kind code	B2
Filing date	Feb 7, 2018
Priority date	Dec 30, 2009
Publication date	Oct 15, 2019
Grant date	Oct 15, 2019

Abstract

A variety of methods and compositions are provided, including methods and compositions for targeted modification of a specific target site in a cell or organism, methods for integrating polynucleotides of interest, methods to assess promoter activity, directly select transformed organisms, minimize or eliminate expression resulting from random integration into the genome of an organism, such as a plant, remove polynucleotides of interest, combine multiple transfer cassettes, invert or excise a polynucleotide, silence a gene, and identify and/or characterize transcriptional regulating regions. The methods involve the introduction of a cell proliferation factor and a double-strand break-inducing enzyme into an organism.

First claim

That which is claimed:

1. A method for modifying a target site of a plant cell, wherein said target site of said plant cell comprises a recognition sequence, and wherein said method comprises:
a) introducing into said plant cell at least one heterologous polynucleotide encoding a Babyboom polypeptide at least 90% identical to SEQ ID NO:39 and expressing said heterologous polynucleotide; wherein said heterologous polynucleotide encoding said Babyboom polypeptide at least 90% identical to SEQ ID NO:39 is operably linked to a promoter active in said plant; and
b) introducing a heterologous polynucleotide encoding a double-strand break-inducing enzyme and expressing said heterologous polynucleotide encoding said double-strand break-inducing enzyme, wherein said double-strand break-inducing enzyme recognizes said recognition sequence and introduces a double-strand break at or near the recognition sequence to produce a modified target site.

2. The method of claim 1 , wherein said promoter operably linked to said heterologous polynucleotide encoding said Babyboom polypeptide at least 90% identical to SEQ ID NO:39 is an oleosin promoter, a ubiquitin promoter, a nopaline synthase promoter, or a In2promoter.

3. The method of claim 1 , wherein said modified target site comprises a deletion, a mutation, a replacement, or an integration of a nucleotide sequence when compared to said target site.

4. The method of claim 1 , wherein said double-strand break-inducing enzyme is selected from the group consisting of an endonuclease, a zinc finger nuclease, a transposase, a topoisomerase, and a site-specific recombinase.

5. The method of claim 4 , wherein said endonuclease comprises a homing endonuclease.

6. The method of claim 5 , wherein said homing endonuclease comprises a modified endonuclease that has been modified to specifically bind said recognition sequence.

7. The method of claim 6 , wherein said modified homing endonuclease is derived from a homing endonuclease selected from the group consisting of I-Sce1, I-Sce11, I-Sce111, I-Sce1V, 1-SceV, 1-SceVI, 1-SceVII, I-Ceu1, 1-CeuAIIP, I-Cre1, 1-Crepsb1P, I-Crepsb1, 1P,I-Crepsb111P,1-Crepsb1VP, I-Tlil, I-Ppol, P1-Psp1, F-Sce1, F-Sce11, F-Suv1, F-Tev1, F-Tev11, I-Ama1, I-Ani1, I-Chu1, I-Cmoe1, I-Cpa1, I-Cpa11, I-Csm1, I-Cvu1, 1-CvuAIP, I-Ddi1, I-Ddi11, I-Dir1, I-Dmo1, I-Hmu1, I-Hmu11, I- HsNIP, I-L1a1, I-Msol, I-Naa1, I-Nan1, 1-Nc11P,1-Ngr1P, I-Nit1, I-Nja1, I-Nsp236IP, I-Pak1, 1-PbolP, 1-PculP, 1 -PcuAI, 1 -PcuVI, 1-Pgr1P, 1-Pob1P, I- Por1, 1-Por11P, 1-Pbp1P, 1-SpBeta1P, I-Sca1, 1-Sex1P, 1-Sne1P, I-Spom1, I- SpomCP,1-Spom1P,1-Spom11P,11-Squ1P, 1- Ssp 68031, 1-SthPhiJP, I- SthPhiST3P, 1-SthPhiSTe 3bP, 1-Tde1P, I-Tev1, I-Tev11, I-Tev111, 1-UarAP, I- UarHGPAIP, 1-UarHGPA 13P, 1-Vin1P, 1-Zbi1P, Pl-Mtu1, PI-MtuHIP P1-MtuHIIP, P1-Pfu1, P1-Pfu11, P1-Pko1, P1Pko11, PI-Rma43812IP, PI-SpBeta1P, P1-Sce1, P1-Tfu1, P1-Tfu11, P1-Thy1, PI-TIiI, and PI-TiiII.

8. The method of claim 1 , wherein said double-strand break-inducing enzyme is a site-specific recombinase and said recognition sequence comprises a first recombination site.

9. The method of claim 8 , wherein said site-specific recombinase is selected from the group consisting of FLP recombinase, Cre, SSV1, R, Gin, lambda Int, phiC31 Int, Tn1721, CinH, ParA, Tn5053, Bxbl, TP907-1, U153, and HK022 Int.

10. The method of claim 8 , wherein said target site further comprises a second recombination site, wherein said target site comprises the following operably linked components: said first recombination site, a nucleic acid sequence, and a second recombination site.

11. The method of claim 10 , wherein said first recombination site is recombinogenic with the second recombination site in the presence of said site-specific recombinase.

12. The method of claim 11 , wherein said nucleic acid sequence is excised or inverted to produce the modified target site.

13. The method of claim 1 , wherein said modified target site comprises an integrated polynucleotide of interest, and wherein said method further comprises introducing into said plant cell a transfer cassette comprising said polynucleotide of interest.

14. The method of claim 13 , wherein said transfer cassette comprises at least a first region having homology to said target site.

15. The method of claim 14 , wherein said transfer cassette comprises in the following order: said first region of homology to said target site, said polynucleotide of interest, and a second region of homology to said target site.

16. The method of claim 1 , said method further comprising selecting cells comprising the modified target site and regenerating a plant having the modified target site.

17. The method of claim 16 , wherein said method further comprises reducing the activity of said Babyboom polypeptide at least 90% identical to SEQ ID NO:39 prior to regenerating a plant having the modified target site.

18. The method of claim 17 , wherein reducing the activity of said Babyboom polypeptide at least 90% identical to SEQ ID NO:39 comprises excising said heterologous polynucleotide encoding said Babyboom polypeptide at least 90% identical to SEQ ID NO:39.

19. The method of claim 18 , wherein said heterologous polynucleotide encoding said Babyboom polypeptide at least 90% identical to SEQ ID NO:39 is flanked by recombination sites, and wherein said method further comprises introducing into said plant cell a site-specific recombinase capable of recognizing and implementing recombination at the recombination sites flanking said heterologous polynucleotide encoding said Babyboom polypeptide at least 90% identical to SEQ ID NO:39, whereby said heterologous polynucleotide encoding said Babyboom polypeptide at least 90% identical to SEQ ID NO:39is excised in the presence of said site-specific recombinase.

20. The method of claim 19 , wherein said introducing said site-specific recombinase capable of recognizing and implementing recombination at the recombination sites flanking said heterologous polynucleotide encoding said Babyboom polypeptide at least 90% identical to SEQ ID NO:39 comprises introducing a heterologous polynucleotide encoding said site-specific recombinase capable of recognizing and implementing recombination at the recombination sites flanking said heterologous polynucleotide encoding said Babyboom polypeptide at least 90% identical to SEQ ID NO:39 and expressing said heterologous polynucleotide encoding said site-specific recombinase capable of recognizing and implementing recombination at the recombination sites flanking said heterologous polynucleotide encoding said Babyboom polypeptide at least 90% identical to SEQ ID NO:39.

21. The method of claim 20 , wherein said heterologous polynucleotide encoding said site-specific recombinase capable of recognizing and implementing recombination at the recombination sites flanking said heterologous polynucleotide encoding said Babyboom polypeptide at least 90% identical to SEQ ID NO:39 is operably linked to an inducible promoter.

22. The method of claim 20 , wherein said heterologous polynucleotide encoding said Babyboom polypeptide at least 90% identical to SEQ ID NO:39 and said heterologous polynucleotide encoding said site-specific recombinase capable of recognizing and implementing recombination at the recombination sites flanking said heterologous polynucleotide encoding said Babyboom polypeptide at least 90% identical to SEQ ID NO:39 are flanked by said recombination sites, whereby said heterologous polynucleotide encoding said Babyb

Assignees

• Pioneer Hi Bred Int

Inventors

• Gordon-Kamm William J
• Lowe Keith S
• Peterson David J
• Scelonge Christopher J
• St Clair Grace M
• Wang bing-bing

Classifications

• C12N15/8213 Primary

  Targeted insertion of genes into the plant genome by homologous recombination · CPC title

• C07K14/415

  from plants · CPC title