# US 2021/0340532 A1 · Thermostable cas9 nucleases

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- Number: US 2021/0340532 A1
- Document ID: US20210340532A1
- Status: Published application
- Kind: A1
- Application number: 16/469,677
- Filed: 2017-08-16
- Published: 2021-11-04
- Earliest claimed: 2016-12-14
- Assignees: Wageningen Universiteit; Stichting voor de Technische Wetenschappen
- Applicants: Wageningen Universiteit; Stichting Voor De Technische Wetenschappen
- Inventors: John Van Der Oost; Richard Van Kranenburg; Elleke Fenna Bosma; Ioannis Mougiakos; Prarthana Mohanraju
- Web page: https://patentgrep.com/patent/US20210340532A1

## CPC

- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/222: {Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity}
- C12N 15/113: 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)}
- C12N 15/902: {using homologous recombination}
- C12N 15/907: {in mammalian cells}
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2330/51: Specially adapted vectors
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Thermostable Cas9 nucleases. 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 20° C. and 100° C. inclusive. The invention further provides isolated nucleic acid molecules encoding said 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 in general, in particular at elevated temperatures and are of particular value in the genetic manipulation of thermophilic organisms; particularly microorganisms.

## Claims

### Claim at position 1

-106. (canceled)

### Claim 107

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 an amino acid sequence of SEQ ID NO: 1 or a sequence of at least 77% identity therewith; and
c) the ribonucleoprotein complex binding, cleaving, marking or modifying the target polynucleotide, and wherein said method is not used in human cells.

### Claim 108 (depends on 107)

The method as claimed in claim 107, wherein the binding, cleaving, marking or modifying occurs at a temperature between 20° C. and 100° C.

### Claim 109 (depends on 107)

The method as claimed in claim 107, wherein the double stranded target polynucleotide comprising the target nucleic acid sequence is cleaved by the Cas protein, preferably wherein said cleavage is DNA cleavage, more preferably which results in a double stranded break in the polynucleotide.

### Claim 110 (depends on 107)

The method as claimed in claim 107, 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.

### Claim 111 (depends on 107)

The method as claimed in claim 107, wherein the PAM sequence comprises at least one sequence selected from the group consisting of 5′-NNNNCNNA-3′ (SEQ ID NO: 47), 5′-NNNNCSAA-3′ (SEQ ID NO: 48) and 5′-NNNNCCAA-3′ (SEQ ID NO: 50).

### Claim 112 (depends on 111)

The method as claimed in claim 111, wherein the binding, cleaving, marking or modifying occurs at a temperature between 20° C. and 70° C.

### Claim 113 (depends on 107)

A method as claimed in claim 107, 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.

### Claim 114 (depends on 107)

The method as claimed in claim 107, wherein the targeting RNA molecule comprises a crRNA and a tracrRNA.

### Claim 115 (depends on 107)

The method as claimed in claim 107, wherein at least one of (a) the length of the at least one targeting RNA molecule is in the range 35-200 nucleotide residues; and (b) the target nucleic acid sequence is from 15 to 32 nucleotide residues in length.

### Claim 116 (depends on 107)

The method as claimed in claim 107, 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 localization sequence, an antibody epitope and an affinity purification tag.

### Claim 117 (depends on 116)

The method as claimed in claim 116, wherein the native activity of the Cas9 nuclease is inactivated and the Cas protein is linked to the at least one functional moiety.

### Claim 118 (depends on 116)

The method as claimed in claim 116, 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.

### Claim 119 (depends on 116)

The method as claimed in claim 116, 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.

### Claim 120 (depends on 107)

The method as claimed in claim 107, wherein said binding, cleaving, marking or modifying occurs in vivo; and optionally further wherein said binding, cleaving, marking or modifying occurs in an organism selected from the group consisting of a thermophilic organism and a mesophilic organism.

### Claim 121 (depends on 107)

The method as claimed in claim 107, 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.

### Claim 122

A transformed non-human 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 an amino acid sequence of SEQ ID NO: 1 or a sequence of at least 77% 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.

### Claim 123 (depends on 122)

The transformed cell as claimed in claim 122, wherein the cell is a prokaryotic cell.

### Claim 124 (depends on 122)

The transformed cell as claimed in claim 122, wherein the Cas protein is expressed from an expression vector.

### Claim 125

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 an amino acid sequence of SEQ ID NO: 1 or a sequence of at least 77% 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′; wherein the nucleoprotein complex is not in a human cell.

### Claim 126 (depends on 125)

The nucleoprotein complex as claimed in claim 125, wherein the nucleoprotein complex is in a prokaryotic cell.