Compositions and methods comprising sequences having meganuclease activity

Patent metadata

Field	Value
Publication number	US-10150956-B2
Application number	US-201815867385-A
Country	US
Kind code	B2
Filing date	Jan 10, 2018
Priority date	May 4, 2012
Publication date	Dec 11, 2018
Grant date	Dec 11, 2018

Abstract

Compositions and methods comprising polynucleotides and polypeptides having meganuclease activity are provided. Further provided are nucleic acid constructs, yeast, plants, plant cells, explants, seeds and grain having the meganuclease sequences. Various methods of employing the meganuclease sequences are provided. Such methods include, for example, methods for producing a meganuclease with increased activity at a wide range of temperatures, methods for producing a yeast, plant, plant cell, explant or seed comprising a meganuclease with increased activity.

First claim

What is claimed:

1. An isolated or recombinant polynucleotide encoding a meganuclease polypeptide, wherein said meganuclease polypeptide has at least 80% sequence identity to SEQ ID NO:283, comprises a lysine (K) at an amino acid position corresponding to position 66 of SEQ ID NO:283, and further comprises:
a) at least one amino acid modification at an amino acid position corresponding to a position of SEQ ID NO: 283 selected from the group consisting of positions 16, 19, 22, 50, 54, 56, 81, 103, 121, 132, 153, 244, 258, 281, 308, 316, 319, and combinations thereof; or,
b) an amino acid sequence having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 of any of the amino acid modification of (a).

2. The isolated or recombinant polynucleotide of claim 1 , wherein said at least one amino acid modification comprises:
a) an isoleucine (I) at a position corresponding to amino acid position 16 in SEQ ID NO: 283;
b) a glycine (G) at a position corresponding to amino acid position 19 in SEQ ID NO: 283;
c) a cysteine (C) at a position corresponding to amino acid position 22 in SEQ ID NO: 283;
d) an arginine (R) or lysine (K) at a position corresponding to amino acid position 50 in SEQ ID NO: 283;
e) an isoleucine (I) at a position corresponding to amino acid position 54 in SEQ ID NO: 283;
f) an aspartic acid (D) at a position corresponding to amino acid position 56 in SEQ ID NO: 283;
g) a lysine (K) at a position corresponding to amino acid position 81 in SEQ ID NO: 283;
h) an asparagine (N) at a position corresponding to amino acid position 103 in SEQ ID NO: 283;
i) a lysine (K) at a position corresponding to amino acid position 121 in SEQ ID NO: 283;
j) an isoleucine (I) at a position corresponding to amino acid position 132 in SEQ ID NO: 283;
k) an aspartic acid (D) at a position corresponding to amino acid position 153 in SEQ ID NO: 283;
l) a glutamic acid (E) at a position corresponding to amino acid position 244 in SEQ ID NO: 283;
m) a serine (S) at a position corresponding to amino acid position 258 in SEQ ID NO: 283;
n) a tyrosine (Y) at a position corresponding to amino acid position 281 in SEQ ID NO: 283;
o) a lysine (K) at a position corresponding to amino acid position 308 in SEQ ID NO: 283;
p) a valine (V) at a position corresponding to amino acid position 316 in SEQ ID NO: 283;
q) an isoleucine (I) at a position corresponding to amino acid position 319 in SEQ ID NO: 283; or
r) any combination of a) to q).

3. The isolated or recombinant polynucleotide of claim 1 , wherein said nucleotide sequence encodes a meganuclease polypeptide, wherein said polypeptide further comprises:
a) an isoleucine (I) or phenylalanine (F) at a position corresponding to amino acid position 16 in SEQ ID NO: 283;
b) a serine (S) or a glycine (G) at a position corresponding to amino acid position 19 in SEQ ID NO: 283;
c) a cysteine (C) or a serine (S) at a position corresponding to amino acid position 22 in SEQ ID NO: 283;
d) an arginine (R) or lysine (K) or glutamine (Q) at a position corresponding to amino acid position 50 in SEQ ID NO: 283;
e) an isoleucine (I) or a phenylalanine (F) at a position corresponding to amino acid position 54 in SEQ ID NO: 283;
f) a leucine (L) or aspartic acid (D) at a position corresponding to amino acid position 56 in SEQ ID NO: 283;
g) a lysine (K) or isoleucine (I) at a position corresponding to amino acid position 81 in SEQ ID NO: 283;
h) a valine (V) or asparagine (N) at a position corresponding to amino acid position 103 in SEQ ID NO: 283;
i) a glycine (G) or lysine (K) at a position corresponding to amino acid position 121 in SEQ ID NO: 283;
j) a valine (V) or isoleucine (I) at a position corresponding to amino acid position 132 in SEQ ID NO: 283;
k) an aspartic acid (D) or a methionine (M) at a position corresponding to amino acid position 153 in SEQ ID NO: 283;
l) a lysine (K) or a glutamic acid (E) at a position corresponding to amino acid position 244 in SEQ ID NO: 283;
m) a serine (S) or a glycine (G) at a position corresponding to amino acid position 258 in SEQ ID NO: 283;
n) a phenylalanine (F) or a tyrosine (Y) at a position corresponding to amino acid position 281 in SEQ ID NO: 283;
o) a glycine (G) or a lysine (K) at a position corresponding to amino acid position 308 in SEQ ID NO: 283;
p) an alanine (A) or a valine (V) at a position corresponding to amino acid position 316 in SEQ ID NO: 283;
q) a valine (V) or an isoleucine (I) at a position corresponding to amino acid position 319 in SEQ ID NO: 1; or
r) any combination of a) to q).

4. The isolated or recombinant polynucleotide of claim 1 , wherein said nucleotide sequence encodes a meganuclease polypeptide selected from the group consisting of SEQ ID NOS: 316, 317, 318, 319, and 320.

5. The isolated or recombinant polynucleotide of claim 1 , wherein said nucleotide sequence encodes a meganuclease polypeptide, wherein the polypeptide is capable of recognizing and cleaving a meganuclease recognition sequence of SEQ ID NO: 281.

6. The isolated or recombinant polynucleotide of claim 1 , wherein said polypeptide has an increased meganuclease activity when compared to a control meganuclease that lacks said amino acid modification.

7. The isolated or recombinant polynucleotide of claim 6 , wherein said control meganuclease is selected from the group of SEQ ID NO: 282 and SEQ ID NO: 283.

8. The isolated or recombinant polynucleotide of claim 6 , wherein the increased meganuclease activity is evidenced by:
a) a higher yeast assay score when compared to the control meganuclease that lacks said amino acid modification; or,
b) a higher target site mutation rate when compared to the control meganuclease that lacks said amino acid modification; or,
c) a higher in-vitro cutting when compared to the control meganuclease that lacks said amino acid modification; or,
d) any combination of (a), (b) and (c).

9. The isolated or recombinant polynucleotide of claim 6 , wherein the increased meganuclease activity is determined at 16° C., 24° C., 28° C., 30° C. or 37° C.

10. A recombinant DNA construct, comprising the isolated or recombinant polynucleotide of claim 1 .

11. A cell comprising at least one polynucleotide of claim 1 or the recombinant DNA construct of claim 10 , wherein said polynucleotide is heterologous to the cell.

12. The cell of claim 11 , wherein said cell is a plant cell.

13. The cell of claim 11 , wherein said plant cell is from a monocot.

14. The cell of claim 11 , wherein said plant cell is from a dicot.

15. A plant comprising a plant cell of claim 12 .

16. A transgenic seed produced by the plant of claim 15 , wherein said transgenic seed comprises the heterologous polynucleotide of claim 1 or the recombinant construct of claim 10 .

17. A method for producing a meganuclease having increased activity over a range of temperatures, the method comprising:
a) producing a variant meganuclease, wherein said variant meganuclease has at least 80% sequence identity to SEQ ID NO:283 and comprises a lysine (K) at an amino acid position corresponding to position 66 of SEQ ID NO:283, by modifying at least one amino acid at an amino acid position corresponding to a position of SEQ ID NO: 283 selected from the group consisting of positions 16, 19, 22, 50, 54, 56, 81, 103, 121, 132, 153, 244, 258, 281, 308, 316, 319, and combinations thereof;
b) screening said variant meganuclease for the ability to cleave a DNA target sequence over a range of temperatures between and including 16° C. to 37° C.; and
c) selecting a variant meganucle

Assignees

• Du Pont
• Pioneer Hi Bred Int
• Du Pont
• Pioneer Hi Bred Iinternational Inc

Inventors

• Bermudez Ericka
• Cigan Andrew Mark
• English James J
• Falco Saverio Carl
• Gao Huirong
• Liu Lu
• Liu Zhan-Bin
• Ong Azalea S
• Svitashev Sergei
• Young Joshua K

Classifications

• C12N15/8213

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

• C12Y301/00

  Hydrolases acting on ester bonds (3.1) · CPC title

• C12N9/22 Primary

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N9/16

  acting on ester bonds (3.1) · CPC title

• C12N15/81

  for yeasts · CPC title