Single-vector type I vectors
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.
1. A cell of a production strain of Escherichia coli ( E. coli ) cells, comprising a first DNA construct wherein:
the first DNA construct comprises a nucleotide sequence encoding a Cas nuclease, wherein the nucleotide sequence is under the control of a promoter for controlling the expression of the Cas nuclease in the production strain cell, wherein
the first DNA construct comprises an origin of replication that is operable in the cell for replication of the construct;
the promoter comprises a nucleotide sequence that is capable of binding to a repressor;
wherein the production strain cell comprises a nucleic acid encoding the repressor;
wherein the promoter is capable of being repressed by the repressor in the production strain;
wherein the cell of the production strain comprises amplified copies of the first DNA construct, wherein the amplified copies of the first DNA construct do not comprise the nucleic acid encoding the repressor,
wherein the nucleic acid encoding the repressor is a chromosomally-integrated sequence or comprised by a second DNA construct; and
wherein the nucleic acid encoding the repressor is capable of expressing the repressor in the cell of the production strain comprising the amplified copies of the first DNA construct;
wherein the cell of the production strain comprises a nucleotide sequence whose expression is inducible to produce phage coat proteins in the cell of the production strain,
wherein the cell of the production strain is capable of packaging the amplified copies of the first DNA construct into phage particles or non-self-replicative transduction particles for introducing the first DNA into a target host cell, and wherein the phage particles or non-self-replicative transduction particles are devoid of a nucleotide sequence encoding the repressor; and
wherein the Cas nuclease is operable with one or more crRNAs or gRNAs to cut target nucleotide sequences in the target host cell, and 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.
2. The cell of claim 1 , wherein the nucleotide sequence that is capable of binding to the repressor is a tetO or lacO.
3. The cell of claim 2 , wherein the promoter is P LtetO-1 , P Llaco-1 , or a repressible homologue thereof.
4. The cell of claim 1 , wherein the repressor is a tetracycline repressor (TetR) or a lac repressor (LacR).
5. The cell of claim 1 , wherein the nucleic acid encoding the repressor is a chromosomally-integrated sequence.
6. The cell of claim 1 , wherein the nucleic acid encoding the repressor is a sequence comprised by an episome.
7. The cell of claim 1 , wherein the first DNA construct is a plasmid or phagemid.
8. The cell of claim 7 , wherein the first DNA construct is a high copy number plasmid or phagemid.
9. The cell of claim 1 , wherein the promoter for controlling the expression of the Cas nuclease in the production strain cell combined with a translation initiation site (TIS) is capable of producing expression of green fluorescent protein (GFP) from a first expression operating unit (EOU) in E. coli strain BW25113 cells with a fluorescence of from 0.5 to 4 times the fluorescence produced in E. coli strain BW25113 cells using a second EOU comprising a P10 promoter having a sequence of SEQ ID NO: 1 combined with a BCD14 TIS having a sequence of SEQ ID NO: 2, wherein the EOUs differ only in their promoter and TIS combinations, wherein each EOU comprises in 5′ to 3′ direction an upstream initiator, the respective promoter, the respective TIS, a nucleotide sequence encoding GFP, a 3′ UTR, a transcription terminator and a downstream insulator.
10. The cell of claim 9 , wherein fluorescence using the first EOU is 0.5 to 2 times the fluorescence using the second EOU.
11. The cell of claim 1 , wherein the cell is capable of growth and propagation sufficient to produce at least 1000 copies of the first DNA construct.
12. The cell of claim 1 , wherein at least 10 5 copies of the first DNA construct can be produced per 10 3 cells of the production strain.
13. The cell of claim 1 , wherein a cell of the production strain is capable of at least 2 or 3 logs of expansion when the first DNA construct is comprised therein.
14. The cell of claim 1 , wherein the Cas nuclease is a Type I Cas nuclease.
15. The cell of claim 1 , wherein the Cas nuclease is a Cas3 nuclease.
16. The cell of claim 15 , wherein the first DNA construct or the cell encodes Cascade proteins that are cognate with the Cas3 nuclease.
17. The cell of claim 1 , wherein the Cas nuclease is a Cas9 nuclease.
18. The cell of claim 1 , wherein the Cas nuclease is a Cpf1 nuclease.
19. The cell of claim 1 , wherein the first DNA construct comprises one or more nucleotide sequences for producing crRNAs or gRNAs that are operable with the Cas nuclease to cut target nucleotide sequences in the target host cell.
20. The cell of claim 1 , wherein the target host cell is comprised by a gut microbiota.
21. The cell of claim 1 , wherein the target host cell is selected from the group consisting of a C. difficile, P. aeruginosa, K. pneumoniae, E. coli, H. pylori, S. pneumoniae and S. aureus cell.