RATIONALLY-DESIGNED MEGANUCLEASES FOR MAIZE GENOME ENGINEERING

• US 20110113509A1
• Filed: 09/10/2010
• Published: 05/12/2011
• Est. Priority Date: 03/11/2008
• Status: Active Grant

• Current Assignee: Precision BioSciences Incorporated
• Original Assignee: Precision BioSciences Incorporated
• Inventors: SMITH, James Jefferson, JANTZ, Derek
• Granted Patent: US 8,338,157 B2
• Time in Patent Office: Days
• US Class Current: 800/278
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

The invention relates to the field of molecular biology and recombinant nucleic acid technology. In particular, the invention relates to a rationally-designed, non-naturally-occurring meganuclease with altered DNA recognition sequence specificity which recognizes and cleaves a unique DNA site in the maize genome. The invention also relates to methods of producing engineered maize plants using such meganucleases.

Claims

1. A rationally-designed meganuclease having the amino acid sequence of SEQ ID NO:

1.

2. A rationally-designed meganuclease having the amino acid sequence of SEQ ID NO:

12.

3. A rationally-designed meganuclease having the amino acid sequence of SEQ ID NO:

13.

4. A rationally-designed meganuclease having at least 85% sequence identity to the amino acid sequence of SEQ ID NO:

1, SEQ ID NO;

12 and/or SEQ ID NO;

13 which recognizes and cleaves the double-stranded DNA sequence of SEQ ID NO;

2 and SEQ ID NO;

3.

5. A method for the production of a recombinant maize plant in which a sequence of interest is integrated the maize genome, the method comprising transforming a maize cell or embryo with:

(a) a first DNA molecule comprising;

a DNA sequence encoding a meganuclease having at least 85% sequence identity to the amino acid sequence of SEQ ID NO;

1, SEQ ID NO;

12 and/or SEQ ID NO;

13 which recognizes and cleaves the double-stranded DNA sequence of SEQ ID NO;

2 and SEQ ID NO;

3; and

a promoter suitable for the expression of such a meganuclease in maize; and

(b) a second DNA molecule comprising a sequence of interest.

7. The method of claim 5 or 6 in which the sequence of interest is flanked on at least one side by the first nine nucleotides from the meganuclease recognition sequence of SEQ ID NO:

2 or SEQ ID NO;

3.

8. The method of claim 5 or 6 wherein the maize cell or embryo is transformed with a molecule comprising the first DNA molecule and the second DNA molecule.

9. The method of claim 5 or 6 wherein the maize cell or embryo is transformed with a molecule comprising the first DNA molecule and the second DNA molecule and wherein SEQ ID NO:

2 or SEQ ID NO;

3 is located between the DNA sequence encoding the meganuclease and the sequence of interest.

6. A method for the production of a recombinant maize plant in which a sequence of interest is integrated the maize genome, the method comprising transforming a maize cell or embryo with:

(a) a first DNA molecule comprising;

a DNA sequence encoding a meganuclease having at least 85% sequence identity to the amino acid sequence of SEQ ID NO;

1, SEQ ID NO;

12 and/or SEQ ID NO;

13 which recognizes and cleaves the double-stranded DNA sequence of SEQ ID NO;

2 and SEQ ID NO;

3; and

a promoter suitable for the expression of such a meganuclease in maize; and

(b) a second DNA molecule comprising;

a sequence of interest flanked on one side by a DNA sequence with homology to 50-900 consecutive base pairs of SEQ ID NO;

10 and flanked on the other side by a DNA sequence with homology to 50-900 consecutive base pairs of SEQ ID NO;

11.

10. A maize plant with a chromosome comprising a composite sequence, the composite sequence comprising:

a sequence of interest;

a first sequence comprising 50-900 base pairs from SEQ ID NO;

10 immediately upstream of the sequence of interest; and

a second sequence comprising 50-900 base pairs from SEQ ID NO;

11 immediately downstream of the sequence of interest.

11. The maize plant of claim 10 wherein the composite sequence is free of retroviral sequences, Agrobacterium tumefaciens sequences and/or transposon sequences.

12. The maize plant of claim 10 with a single composite sequence in said chromosome.

13. The maize plant of claim 10 wherein the 3′

nucleotide of the first sequence corresponds to a nucleotide selected from the group consisting of nucleotides 950-961 of SEQ ID NO;

10.

14. The maize plant of claim 10 wherein the 5′

nucleotide of the second sequence corresponds to a nucleotide selected from the group consisting of nucleotides 1-11 of SEQ ID NO;

11.

15. The maize plant of any one of claims 10-14 wherein the sequence of interest comprises an exogenous gene.

16. A maize plant having a sequence of interest inserted in its chromosome, wherein the sequence of interest is inserted into SEQ ID NO:

2 and SEQ ID NO;

3.

17. The maize plant of claim 16 wherein the sequence of interest is inserted only once into said chromosome.

18. The maize plant of claim 16 or 17 wherein the sequence of interest comprises an exogenous gene.