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US 11,578,333 B2Granted
Single-vector type I vectors
AssigneeSNIPR Biome ApS
InventorsVirginia Martinez, Ruben Vazquez-Uribe, Adam Takos, Eric Van Der Helm, Jasper Clube
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Abstract
The invention relates to the production and use of Cas-encoding sequences and vectors comprising these. Aspects of the invention provide products, vectors, delivery vehicles, uses and methods for producing Cas-encoding sequences in bacterial or archaeal cells.
Claims18
A method for enhancing the yield of amplified copies of a first DNA construct in a population of Escherichia coli (E. coli) production strain cells,
- wherein the first DNA construct comprises a nucleotide sequence encoding a Cas nuclease that is under the control of a promoter for controlling the expression of the Cas nuclease in the production strain cells,
- wherein the promoter comprises a nucleotide sequence that is capable of binding to a repressor;
- wherein production strain cells comprise a nucleic acid sequence encoding the repressor,
- wherein the nucleic acid sequence encoding the repressor is a chromosomally-integrated sequence or comprised by a second DNA construct, and the amplified copies of the first DNA construct do not comprise the nucleic acid sequence encoding the repressor;
- the method comprising culturing the cells to allow replication of the first DNA construct thereby amplifying the first DNA construct in the cells and to allow expression of the repressor in the cells,
- wherein the promoter is repressed by the repressor in the production strain cells,
- whereby the yield of amplified copies of the first DNA construct is enhanced relative to the yield produced using a constitutive promoter having an Anderson Score (AS) of AS≥0.5 to control the expression of the Cas nuclease in the production strain cells,
- wherein the production strain cell does not comprise a crRNA or gRNA operable with the Cas nuclease to target and cut a chromosomal sequence of the production strain cell.
5 more elements
A method of making phage particles or non-self replicative transduction particles comprising amplified copies of a first DNA construct, the method comprising:
- (a) culturing a population of Escherichia coli (E. coli) production strain cells, wherein the production strain cells comprise:
- (i) the first DNA construct, wherein the first DNA construct comprises a nucleotide sequence encoding a Cas nuclease that is under the control of a promoter for controlling the expression of the Cas nuclease in the production strain cells, and wherein the promoter comprises a nucleotide sequence that is capable of binding to a repressor,
- (ii) a nucleic acid sequence encoding the repressor, wherein the nucleic acid sequence encoding the repressor is a chromosomally-integrated sequence or comprised by a second DNA construct, and
- (iii) a nucleotide sequence that is inducible to produce phage coat proteins in the cells;
- wherein the cultured production strain cells express the repressor in the cells and replicate the first DNA construct to produce the amplified copies of the first DNA construct, wherein the amplified copies of the first DNA construct do not comprise the nucleic acid sequence encoding the repressor;
- and
- (b) inducing production of the phage proteins and causing packaging of the amplified DNA into phage particles or non-self-replicative transduction particles, and further isolating the phage or transduction particles,
- wherein the phage particles or non-self-replicative transduction particles are devoid of the nucleic acid sequence encoding the repressor.
5 more elements
Dependencies are inferred from the claim text.