Thermostable Cas9 nucleases

Patent metadata

Field	Value
Publication number	US-11242513-B2
Application number	US-201616469674-A
Country	US
Kind code	B2
Filing date	Dec 14, 2016
Priority date	Dec 14, 2016
Publication date	Feb 8, 2022
Grant date	Feb 8, 2022

Abstract

The present invention relates to the field of genetic engineering and more particularly to nucleic acid editing and genome modification. The present invention provides an isolated Cas protein or polypeptide fragment thereof having an amino acid sequence of SEQ ID NO: 1 or a sequence of at least 77% identity therewith. The Cas protein or polypeptide is capable of binding, cleaving, marking or modifying a double stranded target polynucleotide at a temperature in the range 30° C. and 100° C. inclusive. The invention further provides isolated nucleic acid molecules encoding the Cas9 nucleases, expression vectors and host cells. The invention also provides PAM sequences recognized by the Cas protein or polypeptide, The Cas9 nucleases disclosed herein provide novel tools for genetic engineering at elevated temperatures and are of particular value in the genetic manipulation of thermophilic organisms; particularly microorganisms.

First claim

The invention claimed is:

1. A method of binding, cleaving, marking or modifying a double stranded target polynucleotide, wherein the double stranded target polynucleotide comprises a target nucleic acid strand comprising a target nucleic acid sequence, and a non-target nucleic acid strand comprising a protospacer nucleic acid sequence complementary to the target nucleic acid sequence, said method comprising:
a) designing at least one targeting RNA molecule, wherein the targeting RNA molecule recognizes the target sequence in the target strand, and the non-target strand further comprises a protospacer adjacent motif (PAM) sequence directly adjacent the 3′ end of the protospacer sequence, wherein the PAM sequence comprises 5′-NNNNCNN-3′;
b) forming a ribonucleoprotein complex comprising the targeting RNA molecule and a Cas protein, wherein the isolated Cas protein has the amino acid sequence of SEQ ID NO: 1 or a sequence of at least 89% identity therewith; and
c) the ribonucleoprotein complex binding, cleaving, marking or modifying the target polynucleotide.

2. The method as claimed in claim 1 , wherein the binding, cleaving, marking or modifying occurs at a temperature between 20° C. and 100° C.

3. The method as claimed in claim 1 , wherein the double stranded target polynucleotide comprising the target nucleic acid sequence is cleaved by the Cas protein.

4. The method as claimed in claim 1 , wherein the target polynucleotide comprising the target nucleic acid sequence is double stranded DNA, the Cas protein lacks the ability to cut the double stranded DNA and said method results in gene silencing of the target polynucleotide.

5. The method as claimed in claim 1 , wherein the PAM sequence comprises at least one sequence selected from the group consisting of 5′-NNNNCNNA-3′, 5′-CNNNCNN-3′, 5′-NNNCCNN-3′, 5′-NNCNCNN-3′, 5′-NNNNCCN-3′, 5′-NCNNCNN-3′, 5′-CCCCCCNA-3′ (SEQ ID NO: 10) and 5′-CCCCCCAA-3′ (SEQ ID NO: 11).

6. The method as claimed in claim 1 , wherein the Cas protein is obtainable from a species selected from the group consisting of a bacterium, an archaeon, a virus, a thermophilic bacterium; a Geobacillus sp. and Geobacillus thermodenitrificans.

7. The method as claimed in claim 1 , wherein the targeting RNA molecule comprises a crRNA and a tracrRNA.

8. The method as claimed in claim 1 , wherein the length of the at least one targeting RNA molecule is in the range 35-200 nucleotide residues.

9. The method as claimed in claim 1 , wherein the target nucleic acid sequence is from 15 to 32 nucleotide residues in length.

10. The method as claimed in claim 1 , wherein the Cas protein further comprises at least one functional moiety selected from the group consisting of a helicase, a nuclease, a helicase-nuclease, a DNA methylase, a histone methylase, an acetylase, a phosphatase, a kinase, a transcription activator, a transcription coactivator, a transcription repressor, a DNA binding protein, a DNA structuring protein, a marker protein, a reporter protein, a fluorescent protein, a ligand binding protein, a signal peptide, a subcellular localisation sequence, an antibody epitope and an affinity purification tag.

11. The method as claimed in claim 10 , wherein the native activity of the Cas9 nuclease is inactivated and the Cas protein is linked to the at least one functional moiety.

12. The method as claimed in claim 10 , wherein the double stranded target polynucleotide is dsDNA, the at least one functional moiety is selected from the group consisting of a nuclease and a helicase-nuclease, and the modification is selected from the group consisting of a single-stranded and a double-stranded break at a desired locus.

13. The method as claimed in claim 10 , wherein the double stranded target polynucleotide is dsDNA and the functional moiety is selected from the group consisting of a DNA modifying enzyme, a methylase, an acetylase, a transcription activator and a transcription repressor and the binding, cleaving, marking or modifying results in modification of gene expression.

14. The method as claimed in claim 1 , wherein said binding, cleaving, marking or modifying occurs in vivo.

15. The method as claimed in claim 1 , wherein the binding, cleaving, marking or modifying results in at least one selected from the group consisting of modifying a desired nucleotide sequence at a desired location, deleting a desired nucleotide sequence at a desired location, inserting a desired nucleotide sequence at a desired location, and silencing gene expression at a desired locus.

16. A transformed cell, having a double stranded target polynucleotide comprising a target nucleic acid sequence, wherein the double stranded target polynucleotide comprises a target nucleic acid strand, comprising said target nucleic acid sequence, and a non-target nucleic acid strand, comprising a protospacer nucleic acid sequence complementary to the target nucleic acid sequence, said cell comprising:
a clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) protein having the amino acid sequence of SEQ ID NO: 1 or a sequence of at least 89% identity therewith;
at least one targeting RNA molecule which recognizes the target nucleic acid sequence in the target nucleic acid strand, wherein the non-target strand further comprises a protospacer adjacent motif (PAM) sequence directly adjacent the 3′ end of the protospacer sequence, wherein the PAM sequence comprises 5′-NNNNCNN-3′; and
an expression vector comprising a nucleic acid encoding at least one of said Cas protein and said targeting RNA molecule.

17. The transformed cell as claimed in claim 16 , wherein the cell is a prokaryotic cell.

18. The transformed cell as claimed in claim 16 , wherein the Cas protein is expressed from an expression vector.

19. A nucleoprotein complex comprising a Cas protein, at least one targeting RNA molecule which recognizes a target nucleic acid sequence in a double stranded target polynucleotide, and the target polynucleotide, wherein
the Cas protein has the amino acid sequence of SEQ ID NO: 1 or a sequence of at least 89% identity therewith;
the double stranded target polynucleotide comprises a target nucleic acid strand, comprising said target nucleic acid sequence, and a non-target nucleic acid strand, comprising a protospacer nucleic acid sequence complementary to the target nucleic acid sequence and a protospacer adjacent motif (PAM) sequence directly adjacent the 3′ end of the protospacer sequence, wherein the PAM sequence comprises 5′-NNNNCNN-3′.

20. The nucleoprotein complex as claimed in claim 19 , wherein the nucleoprotein complex is in a prokaryotic cell.

Assignees

• Univ Wageningen
• Stichting Technische Wetenschappen

Inventors

• Van Der Oost John
• Van Kranenburg Richard
• Bosma Elleke Fenna
• Mougiakos Ioannis

Classifications

• C12N15/902

  using homologous recombination · CPC title

• C12N15/102

  Mutagenizing nucleic acids · CPC title

• C12N2310/531

  Stem-loop; Hairpin · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title

• C12N15/113

  Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)} · CPC title