Propagator cells and methods for propagating phage, in particular for delivering crispr-cas components via probiotic organisms
AssigneeSNIPR Technologies Limited
InventorJasper CLUBE
Life of this patent
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Abstract
The invention provides propagator cells and methods for propagating phage and transduction particles.
Claims37
A method of producing a population of phage, wherein the phage are of a first type capable of infecting cells of a first bacterial species or strain (host cells) by binding a cell-surface receptor comprised by bacteria of said species or strain, the method comprising
- (a) Providing a population of second bacterial cells comprising the receptor on the surface thereof, wherein the second cells are of a second species or strain, wherein the second species or strain is different from the first species or strain;
- (b) Infecting the second cells with phage of said first type;
- (c) Propagating the phage in the second cells, thereby producing the population of phage;
- and
- (d) Optionally isolating phage of said population.
A method of producing a population of transduction particles comprising nucleic acid packaged by phage coat proteins, wherein the particles are capable of infecting cells of a first bacterial species or strain (host cells) by binding a cell-surface receptor comprised by bacteria of said species or strain, whereby host cells are transduced with the nucleic acid, the method comprising
- (a) Providing a population of second bacterial cells comprising the receptor on the surface thereof, wherein the second cells are of a second species or strain, wherein the second species or strain is different from the first species or strain, and wherein the second cells comprise DNA that is capable of producing copies of said nucleic acid;
- (b) Infecting the second cells with phage by binding the phage to the receptor comprised by the second bacterial cells;
- (c) Propagating the phage in the second cells, wherein phage coat proteins are produced that package copies of said nucleic acid, thereby producing the population of particles;
- and
- (d) Optionally isolating particles of said population.
The method of any preceding claim, wherein the phage or particles comprise a nucleotide sequence encoding crRNAs (or single guide RNAs) that are operable with Cas in bacteria of said host cell strain or species to form an active CRISPR/Cas system that is capable of targeting one or more protospacer nucleotide sequences, wherein each target sequence is comprised by the genome of said host cells, whereby the crRNAs (or gRNAs) guide Cas in host cells to modify (optionally cut) the target sequence(s), thereby killing host cells or reducing host cell population growth.
The method of any preceding claim, wherein
- (a) the phage or particles comprise a nucleotide sequence encoding crRNAs (or single guide RNAs) that are operable with Cas in bacteria of said host cell strain or species to form an active CR1SPR/Cas system that is capable of targeting one or more protospacer nucleotide sequences, wherein each target sequence is comprised by the genome of said host cells, whereby the crRNAs (or gRNAs) guide Cas in host cells to modify (optionally cut) the target sequence(s), thereby killing host cells or reducing host cell population growth;
- (b) the host and second cells are of the same species (optionally E coli strains);
- and
- (c) the genome of each second bacterial cell does not comprise a said target sequence, wherein the first and second cells are different strains of the same species.
The method of any preceding claim, wherein bacteria of said host species or strain comprise an anti-phage toxin or mechanism for killing or reducing the propagation of phage of said first type or particles that infect host bacteria, wherein the second bacteria do not comprise said toxin or mechanism.
The method of any preceding claim, wherein bacteria of said host species or strain comprise a CRISPR/Cas system that is active for killing or reducing the propagation of phage of said first type or particles that infect host bacteria, wherein the second bacteria do not comprise said system.
The method of any preceding claim, wherein the second bacterial cells are engineered to produce the receptor, wherein wild-type bacteria of said second species or strain do not produce said receptor.
The method of any preceding claim, wherein the phage or particles comprise an origin of replication that is operable in said second cells and in cells of said first species or strain.
The method of any preceding claim, wherein the second cells are E coli cells.
The method of any preceding claim, wherein the first and second cells are of the same species (optionally E coli strains).
The method of any preceding claim, wherein the second cells are cells of a lower hazard category (optionally Hazard Group 1 or 2) compared to cells of the host species or strain (optionally Hazard Group 3 or 4).
The method of any preceding claim, wherein the receptor is selected from lipopolysaccharides, teichoic acids (optionally a ManNAc(β1→4)GlcNAc disaccharide with one to three glycerol phosphates attached to the C4 hydroxyl of the ManNAc residue followed by a long chain of glycerol- or ribitol phosphate repeats), proteins and flagella.
The method of any preceding claim, wherein the receptor comprises an O-antigen of the host cells.
The method of any preceding claim, wherein the phage or particles are operable to express an endolysin or holin in second cells, optionally when phage or particles replicate in second cells.
A cell (propagator cell) for propagating phage or transduction particles comprising nucleic acid packaged by phage coat proteins, wherein the phage or particles are of a first type capable of infecting cells of a first bacterial species or strain (host cells) by binding a cell-surface receptor comprised by bacteria of said species or strain, the propagator cell comprising the receptor on the surface thereof, wherein the propagator cell is of a second species or strain, wherein the second species or strain is different from the first species or strain, whereby the propagator cell is capable of being infected by phage of said first type or said particles for propagation of phage or particles respectively therein.
Dependencies are inferred from the claim text.