Crispr-CAS10 systems and methods for phage genome editing

Inventors

Hatoum, Asma

Assignees

University of Alabama at Birmingham UAB

Abstract

The present disclosure relates to CRISPR-Cas10 systems and methods for phage genome editing.

Core Innovation

The disclosure provides a phage genome editing system for infection of a Staphylococcus bacterial cell by a phage, using a CRISPR-Cas10 system and a donor nucleic acid sequence to introduce a mutated nucleic acid sequence into the phage genome. The system includes a vector comprising a crRNA that can hybridize to a nucleic acid sequence of the phage and a vector comprising a CRISPR-Cas10 system that comprises CRISPR-associated genes csm1/cas10, csm2, csm3, csm4, csm5, csm6, and/or cas6.

The donor nucleic acid sequence includes the mutated nucleic acid sequence to be introduced into the phage, flanked by two nucleic acid sequences containing regions of homology to the phage genome. The disclosure addresses the need for phage genome editing approaches for virulent staphylococcal phages and describes embodiments with endogenous CRISPR-Cas10 and embodiments with heterologous CRISPR-Cas10 on plasmids, enabling targeting of phage nucleic acids by CRISPR-Cas10.

The disclosure further characterizes mutation types that can be introduced into the phage via the donor nucleic acid sequence, including point mutation, insertion mutation, and deletion mutation. It also describes selection constraints and computational assistance for identifying permissable CRISPR-Cas10 protospacers, and reports robust CRISPR-Cas10 immunity across multiple loci and rare recombinant recovery with intended edits in screened plaques.

Claims Coverage

The independent claim defines the overall phage genome editing system, with four principal inventive feature groupings: a Staphylococcus-infectable host, CRISPR-Cas10 targeting via a crRNA-hybridizing vector, a CRISPR-Cas10 gene set, and a donor nucleic acid carrying a mutated phage sequence flanked by regions of homology. No additional independent claims are provided in the partial content; the remaining listed items are dependent claims refining the independent claim.

The claim coverage centers on a phage genome editing system that combines a Staphylococcus bacterial host, a vector carrying a crRNA that hybridizes to a phage nucleic acid sequence, a CRISPR-Cas10 system comprising csm1/cas10 and/or csm2-csm6 and/or cas6 genes, and a donor nucleic acid with a mutated phage sequence flanked by homology regions to the phage genome.

Stated Advantages

Documented Applications