CODON-OPTIMIZED CAS9 ENDONUCLEASE ENCODING POLYNUCLEOTIDE
It was now found that the expression of a nucleotide sequence as described in the method of the invention in a plant cell results in much higher rates of indels compared to those seen in cells transformed with a control nucleic acid molecule. Thus, the invention is directed to codon-optimized Cas9 endonuclease-encoding polynucleotide. Accordingly, the present invention provides a method for modifying a target site in the genome of a plant cell, the method comprising providing one or more guide RNA and a Cas endonuclease to said plant cell, wherein said guide RNA and Cas endonuclease are capable of forming a complex that enables the Cas endonuclease to introduce a double strand break at said target site, and wherein the Cas9 endonuclease is expressed in the plant cell from a polynucleotide comprising an codon-optimized Cas9 endonuclease encoding nucleic acid molecule with a nucleotide sequence selected from the disclosed nucleotide sequences.
1 . A method for modifying a target site in the genome of a plant cell, the method comprising providing one or more guide RNA and a Cas endonuclease to said plant cell, wherein said guide RNA and Cas endonuclease are capable of forming a complex that enables the Cas endonuclease to introduce a double strand break at said target site, and wherein the Cas9 endonuclease is expressed in the plant cell from a polynucleotide comprising a codon-optimized Cas9 endonuclease encoding nucleic acid molecule with a nucleotide sequence selected from the following nucleotide sequences:
a. a nucleotide sequence that in an alignment with the nucleotide sequence depicted in SEQ ID NO. 1 has at the following positions counting from the first nucleotide of the start codon one or more of the following nucleotide combinations:
i. 3018 A and 201 G,
ii. 3018 A and 639 G,
iii. 3018 A and 1248 T,
iv. 4014 A and 201 G,
v. 4014 A and 1329 A,
vi. 4014 A and 1248 T,
vii. 4014 A and 438 G,
viii. 4014 A and 2805 T
ix. 201 G and 2805 T,
x. 201 G and 1248 T,
xi. 201 G and 2460 T, and/or
xii. 201 G and 3648 A,
b. a nucleotide sequence at least 90% identical to SEQ ID NO.: 1; and/or
c. a nucleotide sequence being at least 80% identical to SEQ ID NO. 1 and having in an alignment to the sequence depicted in SEQ ID NO. 1 at the following positions counting from the first nucleotide of the start codon one or more of the following nucleotides:
i. 303 A,
ii. 1029 A,
iii. 1329 A, and/or
iv. 2418 A.
2 . The method of claim 1 , wherein the nucleotide sequence that in an alignment with the nucleotide sequence depicted in SEQ ID NO. 1 has at the following positions counting from the first nucleotide of the start codon one or more of the following nucleotide combinations:
i. 3018 A, 201 G, 639 G and 1248 T,
ii. 4014 A and 201 G, 1329 A,1248 T,438 G, and 2805 T,
iii. 201 G and 2805 T, 1248 T, 2460 T, and 3648 A, and/or
iv. 3018 A, 201 G, 639 G, 1248 T, 4014 A, 1329 A, 438 G, and 2805 T, 2460 T, and 3648 A
3 . The method of claim 1 , wherein the plant is a wheat plant.
4 . The method of claim 1 , wherein the Cas9 endonuclease comprises the polypeptide sequence as shown in SEQ ID NO: 2, or a polypeptide sequence 90% or more identical to SEQ ID NO. 2, or a polypeptide encoded by a nucleotide sequence 90% or more identical to SEQ ID NO. 1.
5 . A polynucleotide molecule encoding a Cas9 endonuclease, wherein the nucleotide sequence of the polynucleotide molecule comprises a nucleotide sequence selected from the following nucleotide sequences:
a. a nucleotide sequence that in an alignment with the nucleotide sequence depicted in SEQ ID NO. 1 has at the following positions counting from the first nucleotide of the start codon one or more of the following nucleotide combinations:
i. 3018 A and 201 G,
ii. 3018 A and 639 G,
iii. 3018 A and 1248 T,
iv. 4014 A and 201 G,
v. 4014 A and 1329 A,
vi. 4014 A and 1248 T,
vii. 4014 A and 438 G,
viii. 4014 A and 2805 T
ix. 201 G and 2805 T,
x. 201 G and 1248 T,
xi. 201 G and 2460 T, and/or
xii. 201 G and 3648 A;
b. a nucleotide sequence at least 90% identical to SEQ ID NO.: 1; and/or
c. a nucleotide sequence being at least 80% identical to SEQ ID NO. 1 and having in an alignment to the sequence depicted in SEQ ID NO. 1 at the following positions counting from the first nucleotide of the start codon one or more of the following nucleotides:
i. 301 A,
ii. 1029 A,
iii. 1329 A, and/or
iv. 2418 A.
6 . The polynucleotide of claim 5 , wherein the nucleotide sequence that in an alignment with the nucleotide sequence depicted in SEQ ID NO. 1 has at the following positions counting from the first nucleotide of the start codon one or more of the following nucleotide combinations:
i. 3018 A, 201 G, 639 G and 1248 T,
ii. 4014 A and 201 G, 1329 A,1248 T,438 G, and 2805 T,
iii. 201 G and 2805 T, 1248 T, 2460 T, and 3648 A, and/or
iv. 3018 A, 201 G, 639 G, 1248 T, 4014 A, 1329 A, 438 G, and 2805 T, 2460 T, and 3648 A.
7 . A method for modifying a target site in the genome of a plant cell, the method comprising providing one or more guide RNAs and a donor DNA to a plant cell having a Cas9 endonuclease, wherein said guide RNA and Cas9 endonuclease are capable of forming a complex that enables the Cas9 endonuclease to introduce a double strand break at said target site, wherein the Cas9 endonuclease is expressed in the plant cell from a polynucleotide comprising the polynucleotide of claim 5 , and wherein said donor DNA comprises a polynucleotide of interest.
8 . A method for modifying a target site in the genome of a wheat cell, the method comprising:
a) providing to a wheat cell one or more guide RNA and a Cas 9 endonuclease encoding sequence, wherein said guide RNA and a Cas 9 endonuclease expressed from said Cas 9 endonuclease encoding sequence are capable of forming a complex that enables the Cas endonuclease to introduce a double strand break at said target site; and,
b) identifying at least one wheat cell that has a modification at said target, wherein the modification includes at least one deletion or substitution of one or more nucleotides in said target site;
and wherein the Cas 9 endonuclease encoding sequence that comprises the polynucleotide sequence of the polynucleotide of claim 5 .
9 . A plant, host cell, a plant cell, a plant organ, or a plant cell compartment comprising a recombinant DNA construct, said recombinant DNA construct comprising a promoter operably linked to a codon-optimized nucleotide sequence encoding a Cas9 endonuclease, wherein said Cas9 endonuclease is capable of binding to and creating a double strand break in a genomic target sequence said plant genome, and wherein the Cas 9 endonuclease encoding sequence that comprises the polynucleotide sequence of the polynucleotide of claim 5 .
10 . A plant, a host cell, a plant cell, a plant organ, or a plant cell compartment comprising a recombinant DNA construct and one or more guide RNA, wherein said recombinant DNA construct comprises a promoter operably linked to a codon-optimized nucleotide sequence encoding a Cas9 endonuclease, wherein said Cas9 endonuclease and guide RNA are capable of forming a complex and creating a double strand break in a genomic target sequence in said plant genome and wherein the Cas 9 endonuclease encoding sequence that comprises the polynucleotide sequence of the polynucleotide of claim 5 .
11 . A recombinant DNA construct comprising a promoter operably linked to a codon-optimized nucleotide sequence encoding a Cas9 endonuclease, wherein said Cas9 endonuclease is capable of binding to and creating a double strand break in a genomic target sequence of said plant genome, wherein the Cas 9 endonuclease encoding sequence comprising the polynucleotide sequence of the polynucleotide of claim 5 .
12 . A recombinant DNA construct comprising a promoter operably linked to a nucleotide sequence expressing a guide RNA, wherein said guide RNA is capable of forming a complex with a Cas9 endonuclease, and wherein said complex is capable of binding to and creating a double strand break in a genomic target sequence said plant genome, and wherein the Cas 9 endonuclease is expressed from a polynucleotide that comprises the polynucleotide sequence of the polynucleotide of claim 5 .
13 . A method for editing a nucleotide sequence in the genome of a cell, the method comprising providing one or more guide RNA, a Cas endonuclease, and optionally a polynucleotide modification template, to a cell, wherein said guide RNA and Cas endonuclease are capable of forming a complex that enables the Cas endonuclease to introduce a double strand break at a target site in the genome of said cell, wherein said polynucleotide modification template comprises at least one nucleotide modification of said nucleotide sequence, and wherein the Cas 9 endonuclease is expressed from a polynucleotide that comprises the polynucleotide sequence of the polynucleotide of claim 5 .
14 . The method of claim 13 , wherein the nucleotide sequence in the genome of a cell is selected from the group consisting of a promoter sequence, a terminator sequence, a regulatory element sequence, a splice site, a coding sequence, a polyubiquitination site, an intron site and an intron enhancing motif.
15 . (canceled)
16 . The polynucleotide molecule of claim 5 , wherein the nucleotide sequence molecule encoding the Cas9 endonuclease further comprises one or more NLS sequence.
17 . The polynucleotide molecule of claim 5 , wherein the Cas9 nuclease is a nickase having if aligned with the Cas9 polypeptide sequence depicted in SEQ ID NO. 2, and/or a D to A mutation at amino acid position 10 and/or a H to A amino acid mutation at position 840, or is a dead nuclease having a R to A mutation at amino acid position 70, and/or a D to A mutation at amino acid position 10 and/or a H to A mutation at amino acid position 840, or having one or more of the mutations as shown in FIG. 1 .
18 . The polynucleotide molecule of claim 5 , wherein the Cas9 nuclease is active or inactive, and is fused to another polypeptide.
19 . The polynucleotide molecule of claim 5 , wherein the Cas9 nuclease is inactive, and is fused to transcription activation or repression effectors, epigenetic factors, such as histone-modifying/DNA methylation enzymes, fluorescent proteins for imaging of specific genomic loci, cytosine or adenine deaminases for precisely altering DNA bases.
20 . The polynucleotide molecule of claim 5 , wherein the Cas9 nuclease is a nickase and is fused to a reverse transcriptase.
21 . The polynucleotide molecule of claim 16 , wherein the one or more NLS sequence is fused to the 5′ terminus and/or is fused to the 3′ terminus of the sequence encoding the Cas9 endonuclease.