Thermostable Cas9 nucleases

J. van der Oost (Inventor), R. van Kranenburg (Inventor), E.F. Bosma (Inventor), I. Mougiakos (Inventor), P. Mohanraju (Inventor)

Research output: PatentOther research output

Abstract

A polynucleotide encoding a ThermoCas9protein from Geobacillus thermodenitrificans and a constitutive promoter are used to engineer eukaryotic cells, e.g. fungi, yeast or algae, so that the ThermoCas9 endonuclease is integrated and expressed from the genome of the cell. Then, a second expression plasmid is used to transfect these ThermoCas9 expressing cells, the second plasmid containing an inducible promoter and a polynucleotide encoding a guide RNA. The guide RNA combines with the ThermoCas9 to provide the targeted endonuclease activity to cleave the cell DNA at a desired locus or gene of interest. A repair-oligo is also provided to the cell whereby following DNA cleavage, homologous recombination takes place in the cell with the repair-oligo so that either a deletion or substitution of nucleotides in the locus or gene of interest is achieved. Expression vectors and methods of using the vectors to achieve ThermoCas9 mediated gene editing are described whereby higher temperatures, e.g. greater than 30 °C, are used.
Original languageEnglish
Patent numberWO2018109101
Priority date14/12/16
Publication statusPublished - 21 Jun 2018

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Micrococcal Nuclease
Guide RNA
Polynucleotides
Endonucleases
Plasmids
Geobacillus
DNA Cleavage
Homologous Recombination
Eukaryotic Cells
Genes
Fungi
Nucleotides
Yeasts
Genome
Temperature
DNA

Cite this

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title = "Thermostable Cas9 nucleases",
abstract = "A polynucleotide encoding a ThermoCas9protein from Geobacillus thermodenitrificans and a constitutive promoter are used to engineer eukaryotic cells, e.g. fungi, yeast or algae, so that the ThermoCas9 endonuclease is integrated and expressed from the genome of the cell. Then, a second expression plasmid is used to transfect these ThermoCas9 expressing cells, the second plasmid containing an inducible promoter and a polynucleotide encoding a guide RNA. The guide RNA combines with the ThermoCas9 to provide the targeted endonuclease activity to cleave the cell DNA at a desired locus or gene of interest. A repair-oligo is also provided to the cell whereby following DNA cleavage, homologous recombination takes place in the cell with the repair-oligo so that either a deletion or substitution of nucleotides in the locus or gene of interest is achieved. Expression vectors and methods of using the vectors to achieve ThermoCas9 mediated gene editing are described whereby higher temperatures, e.g. greater than 30 °C, are used.",
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Thermostable Cas9 nucleases. / van der Oost, J. (Inventor); van Kranenburg, R. (Inventor); Bosma, E.F. (Inventor); Mougiakos, I. (Inventor); Mohanraju, P. (Inventor).

Patent No.: WO2018109101.

Research output: PatentOther research output

TY - PAT

T1 - Thermostable Cas9 nucleases

AU - van der Oost, J.

AU - van Kranenburg, R.

AU - Bosma, E.F.

AU - Mougiakos, I.

AU - Mohanraju, P.

PY - 2018/6/21

Y1 - 2018/6/21

N2 - A polynucleotide encoding a ThermoCas9protein from Geobacillus thermodenitrificans and a constitutive promoter are used to engineer eukaryotic cells, e.g. fungi, yeast or algae, so that the ThermoCas9 endonuclease is integrated and expressed from the genome of the cell. Then, a second expression plasmid is used to transfect these ThermoCas9 expressing cells, the second plasmid containing an inducible promoter and a polynucleotide encoding a guide RNA. The guide RNA combines with the ThermoCas9 to provide the targeted endonuclease activity to cleave the cell DNA at a desired locus or gene of interest. A repair-oligo is also provided to the cell whereby following DNA cleavage, homologous recombination takes place in the cell with the repair-oligo so that either a deletion or substitution of nucleotides in the locus or gene of interest is achieved. Expression vectors and methods of using the vectors to achieve ThermoCas9 mediated gene editing are described whereby higher temperatures, e.g. greater than 30 °C, are used.

AB - A polynucleotide encoding a ThermoCas9protein from Geobacillus thermodenitrificans and a constitutive promoter are used to engineer eukaryotic cells, e.g. fungi, yeast or algae, so that the ThermoCas9 endonuclease is integrated and expressed from the genome of the cell. Then, a second expression plasmid is used to transfect these ThermoCas9 expressing cells, the second plasmid containing an inducible promoter and a polynucleotide encoding a guide RNA. The guide RNA combines with the ThermoCas9 to provide the targeted endonuclease activity to cleave the cell DNA at a desired locus or gene of interest. A repair-oligo is also provided to the cell whereby following DNA cleavage, homologous recombination takes place in the cell with the repair-oligo so that either a deletion or substitution of nucleotides in the locus or gene of interest is achieved. Expression vectors and methods of using the vectors to achieve ThermoCas9 mediated gene editing are described whereby higher temperatures, e.g. greater than 30 °C, are used.

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