Single-vector type I vectors
Inventors
MARTINEZ, Virginia • VAZQUEZ-URIBE, Ruben • TAKOS, Adam • VAN DER HELM, Eric • Clube, Jasper
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Assignees
MemberSNIPR BiomeSNIPR BiomeSNIPR Biome develops precision medicines for infections and diseases linked to antimicrobial resistance and microbiome dysbiosis, using CRISPR-based gene editing to selectively target pathogenic bacteria while sparing the rest of the microbial community. The company applies programmable CRISPR platforms, delivered by engineered phages or probiotics, to address difficult-to-treat infections and modulate the human microbiome. Its programs span discovery through Phase 2 clinical trials, with a significant patent portfolio supporting these innovations.
SNIPR Biome develops precision medicines for infections and diseases linked to antimicrobial resistance and microbiome dysbiosis, using CRISPR-based gene editing to selectively target pathogenic bacteria while sparing the rest of the microbial community. The company applies programmable CRISPR platforms, delivered by engineered phages or probiotics, to address difficult-to-treat infections and modulate the human microbiome. Its programs span discovery through Phase 2 clinical trials, with a significant patent portfolio supporting these innovations.
Publication Number
US-11578333-B2
Publication Date
2023-02-14
Expiration Date
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.
Core Innovation
The described subject matter addresses a need to enhance the yield of amplified copies of a first DNA construct in Escherichia coli production strain cells. In this approach, the first DNA construct comprises a nucleotide sequence encoding a Cas nuclease under the control of a promoter capable of binding to a repressor, and the production strain cells comprise a nucleic acid sequence encoding the repressor expressed during culturing while amplifying the first DNA construct.
The production strain cells are configured such that the amplified copies of the first DNA construct do not comprise the nucleic acid sequence encoding the repressor. The promoter is repressed by the repressor in the production strain cells, and the yield of amplified copies is enhanced relative to a constitutive promoter having an Anderson Score (AS) of AS≤0.5. A further safeguard is specified so that 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.
A related aspect describes making phage particles or non-self replicative transduction particles by culturing Escherichia coli production strain cells to amplify a first DNA construct. The production strain cells comprise a first DNA construct encoding a Cas nuclease under a promoter capable of binding to a repressor, a nucleic acid sequence encoding the repressor that is chromosomally integrated or comprised by a second DNA construct, and a nucleotide sequence inducible to produce phage coat proteins.
After expression of the repressor and replication of the first DNA construct, inducing production of phage coat proteins causes packaging of amplified DNA into phage particles or non-self replicative transduction particles, which are devoid of the nucleic acid sequence encoding the repressor. The described content also includes a requirement that the production strain cell does not comprise operable crRNA or gRNA that could target and cut a chromosomal sequence of the production strain cell.
Claims Coverage
The provided partial content includes two independent claims. Across these, the main inventive features center on repressor-controlled Cas nuclease expression during DNA amplification with an Anderson Score benchmark and a self-targeting exclusion, and packaging of amplified DNA into phage or non-self replicative transduction particles such that the particles are devoid of the repressor sequence.
Repressor-controlled Cas nuclease expression during amplification with no repressor in amplified copies
A method for enhancing the yield of amplified copies of a first DNA construct in Escherichia coli production strain cells, where the first DNA construct encodes a Cas nuclease under a promoter capable of binding to a repressor, and the production strain cells comprise a nucleic acid sequence encoding the repressor expressed in the cells, while the amplified copies of the first DNA construct do not comprise the nucleic acid sequence encoding the repressor and the promoter is repressed by the repressor during culturing.
Self-targeting exclusion for production strain
A method 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.
Yield enhancement relative to an Anderson Score constitutive promoter
A method wherein the yield of amplified copies is enhanced relative to the yield produced using a constitutive promoter having an Anderson Score (AS) of AS≤0.5 to control expression of the Cas nuclease in the production strain cells.
Packaging amplified DNA into particles devoid of repressor sequence
A method of making phage particles or non-self replicative transduction particles by culturing Escherichia coli production strain cells comprising a first DNA construct encoding a Cas nuclease under a repressor-binding promoter, a nucleic acid sequence encoding the repressor (chromosomally integrated or comprised by a second DNA construct), and a nucleotide sequence inducible to produce phage coat proteins, wherein the amplified copies do not comprise the nucleic acid sequence encoding the repressor and inducing production of phage proteins causes packaging of the amplified DNA into phage particles or non-self replicative transduction particles that are devoid of the nucleic acid sequence encoding the repressor.
Across the independent claims, the key inventive theme is repressor-mediated control of Cas nuclease expression during DNA amplification, combined with an explicit requirement that the production strain does not contain operable crRNA/gRNA for self-chromosomal cutting, and a production and induction scheme that yields particles containing amplified DNA while excluding the repressor sequence.
Stated Advantages
Enhances the yield of amplified copies of a first DNA construct relative to a constitutive promoter having an Anderson Score (AS) of AS≤0.5.
Documented Applications
Making phage particles or non-self replicative transduction particles comprising amplified copies of a first DNA construct, followed by isolation of the particles.
Formulating the particles into a pharmaceutical composition for administration to a human or animal subject to treat or reduce the risk of a disease or condition.
Using infective particles to transduce target host cells in a subject, where the first DNA construct expresses a Cas nuclease operable with one or more crRNAs or gRNAs to modify a target sequence in the target host cells.
Cas-mediated cutting of a target sequence leading to killing of target host cells, thereby treating or reducing the risk of a disease or condition.
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