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AssigneeDanisco US Inc.
InventorsRyan L. FRISCH, Stacey Irene Robida STUBBS, Wonchul SUH, Derek Joseph ZIMMER
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Methods and compositions are provided for integrating donor DNA sequences into the genome of a Bacillus sp. cell without the integration of a selectable marker into said genome. The methods employ a linear recombinant DNA construct comprising a donor DNA flanked by long homology arms (each of at least 1000 nucleotides in length) in combination with a recombinant DNA construct encoding a Cas9 endonuclease and a guide RNA, for the introduction of a guide RNA/Cas endonuclease into a Bacillus sp. cell, and as such providing a highly effective system for integrating donor DNA sequences into the genome of said Bacillus sp. cell, without the need to integrate a selectable marker in the genome of said Bacillus sp. cell.
A method of integrating a donor DNA sequence into a target site on the genome of a Bacillus sp. cell without the integration of a selectable marker into said genome, the method comprising simultaneously introducing at least a linear recombinant DNA construct and a circular recombinant DNA construct into a Bacillus sp. cell, wherein said linear recombinant DNA construct comprises a donor DNA sequence, wherein said donor DNA sequence is flanked by an upstream homology arm (HR1) and a downstream arm (HR2), wherein each homology arm is greater than 1000 nucleotides in length, wherein said circular recombinant DNA construct comprises a DNA sequence encoding a guide RNA and a constitutive promoter operably linked to a nucleotide sequence encoding a Cas endonuclease, wherein said Cas9 endonuclease introduces a double-strand break at or near a target site in the genome of said Bacillus sp. cell.
A method of integrating multiple copies of a gene of interest into the genome of a Bacillus sp. cell without the integration of a selectable marker into said genome, the method comprising simultaneously introducing at least a linear recombinant DNA construct and a circular recombinant DNA construct into a Bacillus sp. cell, wherein said linear recombinant DNA construct comprises a donor DNA sequence flanked by an upstream homology arm (HR1) and a downstream arm (HR2), wherein said donor DNA comprises multiple copies of said gene of interest, wherein each homology arm is greater than 1000 nucleotides in length, wherein said circular recombinant DNA construct comprises a DNA sequence encoding a guide RNA and a constitutive promoter operably linked to a nucleotide sequence encoding a Cas endonuclease, wherein said Cas9 endonuclease introduces a double-strand break at or near a target site in the genome of said Bacillus cell.
Dependencies are inferred from the claim text.