Purac Biochem Bv

Stichting Voor De Technische Wetenschappen

Wageningen Universiteit

Richard Van Kranenburg

John Van Der Oost

Ioannis Mougiakos

Elleke Fenna Bosma

Prarthana Mohanraju

Mariska Van Hartskamp

Eelco Anthonius Johannes Heintz

Esther Johanna Geertruda Van Mullekom

Jurgen Snelders

Marinus Petrus Machielsen

Anna Verhoef

Ms Esther Johanna Geertruda Van Mullekom

A polynucleotide encoding a ThermoCas9 protein fromand 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.

Thermostable Cas9 nucleases

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.

Thermostable CAS9 nucleases

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 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 in general, in particular at elevated temperatures and are of particular value in the genetic manipulation of thermophilic organisms; particularly microorganisms.

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&#xb0; C. and 100&#xb0; 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 in general, in particular at elevated temperatures and are of particular value in the genetic manipulation of thermophilic organisms; particularly microorganisms.

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&#xb0; C. and 100&#xb0; 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.

The invention relates to a genetically engineered thermophilic bacterial cell that is facultative anaerobic comprising: a) inactivation or deletion of the endogenous (S)-lactate dehydrogenase gene; b) introduction of a (R)-lactate dehydrogenase gene; c) inactivation or deletion of the endogenous pyruvate formate lyase A and/or B gene.

Genetically modified (R)-lactic acid producing thermophilic bacteria

A genetically engineered thermophilic bacterial cell that is facultative anaerobic and (S)-lactic acid producing including inactivation or deletion of the endogenous methylglyoxal synthase gene mgsA.

Genetic modification of (S)-lactic acid producing thermophilic bacteria

Disclosed herein is a genetic modification of moderately thermophilicspecies that are facultative anaerobic and homolactic. The method includes introducing DNA cloned in a thermosensitive plasmid system containing a pSH71 replicon or a homologue thereof into cells of a moderately thermophilicspecies that is facultative anaerobic and homolactic; culturing the cells on a selective medium at a permissive temperature to select transformed cells; culturing the transformed cells on a selective medium at a non-permissive temperature to select transformed cells capable of growing on the selective medium at the non-permissive temperature. The method can modify the Bacilli for R-lactic acid production, production of other organic acids than lactic acid, alcohol, enzymes, amino acids, and vitamins. Thespecies may be modified by replacing the S-lactate dehydrogenase gene by a DNA construct including a DNA sequence encoding R-lactate dehydrogenase.

Method of preparation of a compound using a genetically modified homolactic thermophilic bacilli

A method for the construction of a moderately thermophilicstrain capable of utilizing sucrose as a carbon source includes the transformation of a parent moderately thermophilicstrain not capable of utilizing sucrose as a carbon source with a polynucleotide comprising a DNA sequence that encodes a polypeptide having sucrose-specific phosphotransferase activity and having i) an amino acid sequence of SEQ ID NO:1 or ii) an amino acid sequence with an identity of at least 70% to the sequence of SEQ ID NO:1 and/or comprising a DNA sequence that encodes a polypeptide having sucrose-6-phosphate hydrolase activity and having iii) an amino acid sequence of SEQ ID NO:2 or iv) an amino acid sequence with an identity of at least 70% to the sequence of SEQ ID NO:2.

Growing community of inventors

Gorinchem, Netherlands

Richard Van Kranenburg