Genetic engineering of bacteriophages using crispr-cas13a
AssigneeThe Regents of the University of California
InventorsJoseph Bondy-Denomy, Jingwen Guan, Senen Mendoza
Life of this patent
Estimating the term…
Abstract
The present disclosure provides methods and compositions for modifying the genomes of bacteriophages, in particular by integrating a coding sequence for an anti-CRISPR protein into the bacteriophage genome together with a desired modification such as a deletion, insertion, or nucleotide substitution, and selecting for bacteriophages with the integrated anti-CRISPR coding sequence by introducing the phage into a counter selection strain comprising an RNA-targeting CRISPR-Cas system.
Claims30
A method of introducing a genetic modification at a locus within the genome of a bacteriophage, the method comprising:
- i) providing a first population of bacterial cells comprising a polynucleotide and the bacteriophage, wherein the polynucleotide comprises a coding sequence for an anti-CRISPR protein that inhibits an RNA-targeting CRISPR-Cas system in the bacterial cells, flanked by two homology arms comprising nucleotide sequence homology to the bacteriophage locus;
- ii) culturing the bacterial cells under conditions conducive to homologous recombination between the polynucleotide and the bacteriophage genome, and obtaining a lysate from the cultured cells;
- iii) infecting a second population of bacterial cells comprising the RNA-targeting CRISPR-Cas system with the lysate;
- and
- iv) isolating modified bacteriophage from the contacted population of CRISPR bacterial cells.
A modified bacteriophage comprising a coding sequence for an anti-CRISPR protein integrated into the bacteriophage genome at a locus that is not essential for phage replication, wherein the anti-CRISPR protein can inhibit an RNA-targeting CRISPR-Cas system, and wherein the locus also comprises a deletion, insertion, or mutation relative to an otherwise equivalent, non-modified bacteriophage.
A bacterial cell comprising a polynucleotide and a bacteriophage, wherein the polynucleotide comprises a coding sequence for an anti-CRISPR protein that inhibits an RNA-targeting CRISPR-Cas system, flanked by two homology arms comprising substantial nucleotide sequence identity to a locus within the genome of the bacteriophage.
Dependencies are inferred from the claim text.