Crispr-CAS10 systems and methods for phage genome editing
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Publication Number
US-11453892-B2
Publication Date
2022-09-27
Expiration Date
Abstract
The present disclosure relates to CRISPR-Cas10 systems and methods for phage genome editing.
Core Innovation
The invention provides a phage genome editing system that uses a Staphylococcus bacterial cell that can be infected by a phage. The system includes a vector comprising a crRNA that can hybridize to a nucleic acid sequence of the phage and a vector comprising a donor nucleic acid sequence carrying a mutated nucleic acid sequence to be introduced into the phage.
The donor nucleic acid sequence includes the mutated nucleic acid sequence flanked by two nucleic acid sequences containing regions of homology to the phage genome. This configuration supports introducing the intended mutated nucleic acid sequence into the phage following nucleic acid targeting by the crRNA, and the described framework is specifically tied to CRISPR-Cas10, including Cas10-Csm effector complex components.
The described system is used in Staphylococcus with configurations that include endogenous CRISPR-Cas10-containing hosts and configurations in which endogenous CRISPR sequences are deleted or where CRISPR-less strains are used. The described edits include point mutations, insertion mutations, and deletion mutations within or relative to protospacer and other phage genome positions, using donor sequences with homology regions to drive editing of virulent lytic phages.
Claims Coverage
The provided independent claim includes 3 inventive components: a phage-infectable Staphylococcus bacterial cell, a crRNA-vector that hybridizes to a phage nucleic acid sequence, and a donor-nucleic-acid vector that carries a mutated phage sequence flanked by regions of homology. The independent claim is refined by multiple dependent claims specifying host configuration, phage class, and donor mutation categories.
Phage-infectable Staphylococcus bacterial cell
A Staphylococcus bacterial cell that can be infected by a phage.
Vector with crRNA hybridizing to phage nucleic acid
A vector comprising a crRNA that can hybridize to a nucleic acid sequence of the phage.
Donor nucleic acid with mutated sequence flanked by homology regions
A vector comprising a donor nucleic acid sequence, wherein the donor nucleic acid sequence comprises a mutated nucleic acid sequence to be introduced into the phage, flanked by two nucleic acid sequences containing regions of homology to the phage genome.
Endogenous CRISPR-Cas10-containing host cell configuration
The system is configured such that the Staphylococcus bacterial cell contains an endogenous CRISPR-Cas10 system.
Deletion of endogenous CRISPR sequences in the host cell
The system includes Staphylococcus bacterial cells with their endogenous CRISPR sequences deleted.
Lytic phage targeting
The phage is a lytic phage.
Donor payload mutation categories
The system uses a mutated nucleic acid sequence chosen from at least one point mutation, an insertion mutation, or a deletion mutation.
Across the independent claim and its refinements, the core claim scope centers on editing a phage genome using a Staphylococcus cell, a crRNA-vector targeting a phage nucleic acid sequence, and a donor-nucleic-acid vector that delivers a mutated phage sequence flanked by regions of homology; dependent claims further define host CRISPR-Cas10 content or deletion, restrict the phage to lytic phages, and constrain the donor mutation types to point, insertion, or deletion mutations.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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