Selection marker free methods for modifying the genome of bacillus and compositions thereof
AssigneeDanisco US Inc.
InventorsFelipe Oseas Bendezu, Stacey Irene Robida Stubbs
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Abstract
Methods and compositions are provided for modifying the genome of Bacillus sp. cells without the use of a selectable marker and without the use of a guided Cas endonuclease. The disclosure includes methods for integrating donor DNA sequences into the genome of a Bacillus sp. cell without the use of a selectable marker and without the use of Cas endonucleases into said genome, as well as methods for deleting genes of interest and/or providing point mutations into the genome of Bacillus sp. cells.
Claims17
A method for integrating a donor DNA into the genome of a Bacillus sp. cell without introducing a nuclease that introduces a double strand break into the genome, and without introducing any circular recombinant DNA, the method comprising providing a population of competent Bacillus sp. cells and introducing only a linear DNA construct into at least one Bacillus sp. cell of said population of cells, wherein said DNA construct comprises a donor DNA flanked by an upstream homology arm (HR1) and a downstream homology arm (HR2), wherein each homology arm is at least 900 nucleotides in length, wherein said DNA construct does not comprise a selectable marker and no selection agent is applied during or after transformation.
A method for deleting a nucleotide sequence in the genome of a Bacillus sp. cell without introducing a nuclease that introduces a double strand break into the genome, and without introducing any circular recombinant DNA, the method comprising providing a population of competent Bacillus sp. cells and introducing only a linear DNA construct into at least one Bacillus sp. cell of said population of cells, wherein said DNA construct comprises an upstream homology arm (HR1) and a downstream homology arm (HR2), wherein each homology arm is at least 900 nucleotides in length and has sequence homology to a genomic DNA region flanking said nucleotide sequence to be deleted, wherein said DNA construct does not comprise a selectable marker and no selection agent is applied during or after transformation.
A method for introducing a mutation in the genome of a Bacillus sp. cell without introducing a nuclease that introduces a double strand break into the genome, and without introducing any circular recombinant DNA, the method comprising providing a population of competent Bacillus sp. cells and introducing only a linear DNA construct into at least one Bacillus sp. cell of said population of cells, wherein said DNA construct comprises a nucleotide sequence having the desired mutation flanked by an up stream homology arm (HR1) and a downstream homology arm (HR2), and wherein each homology arm is at least 900 nucleotides in length, wherein said DNA construct does not comprise a selectable marker and no selection agent is applied during or after transformation.
Dependencies are inferred from the claim text.