Phage engineering: protection by circularized intermediate

Patent metadata

Field	Value
Publication number	US-11535843-B2
Application number	US-201815994002-A
Country	US
Kind code	B2
Filing date	May 31, 2018
Priority date	Jun 1, 2017
Publication date	Dec 27, 2022
Grant date	Dec 27, 2022

Abstract

The present disclosure provides methods of generating recombinant bacteriophage genomes. Specifically, the present technology provides methods of integrating a heterologous nucleic acid sequence into a linear bacteriophage DNA genome, and isolating recombinant bacteriophages that express the heterologous nucleic acid sequence.

First claim

The invention claimed is:

1. A method for integrating a heterologous nucleic acid into a terminally redundant linear bacteriophage DNA genome comprising:
(a) cleaving a first site at the 5′ end of a plurality of terminally redundant linear bacteriophage DNA genomes and a second site at the 3′ end of the plurality of terminally redundant linear bacteriophage DNA genomes with a CRISPR enzyme in vitro, wherein the plurality of terminally redundant linear bacteriophage DNA genomes is present in an isolated sample;
(b) recombining in vitro the cleaved plurality of terminally redundant linear bacteriophage DNA genomes with the heterologous nucleic acid in the presence of a recombination system, wherein the heterologous nucleic acid comprises a 3′ flanking region and a 5′ flanking region that are homologous to the 5′ and 3′ ends of the cleaved plurality of terminally redundant linear bacteriophage DNA genomes respectively, thereby generating a plurality of circularized recombinant bacteriophage DNA genomes; and
(c) enriching the plurality of circularized recombinant bacteriophage DNA genomes by incubating the sample with at least one exonuclease, wherein the heterologous nucleic acid encodes a non-endogenous protein, wherein the plurality of circularized recombinant bacteriophage genomes gives rise to recombinant bacteriophage particles that produce functionally active non-endogenous protein when transformed into a bacterial host cell, wherein the CRISPR enzyme is Cas9, and wherein the plurality of terminally redundant linear bacteriophage DNA genomes are selected from the group consisting of Klebsiella phage K11, Enterobacteria phage T7, and K15 phage.

2. The method of claim 1 , further comprising propagating the plurality of circularized recombinant bacteriophage DNA genomes in a non-natural bacterial host.

3. The method of claim 1 , wherein the at least one exonuclease is selected from the group consisting of Lambda exonuclease, Exonuclease III, RecBCD, Exonuclease VIII truncated, T5 exonuclease, and T7 exonuclease.

4. The method of claim 1 , wherein the recombination system comprises a 5′-3′ exonuclease, a DNA polymerase, and a DNA ligase.

5. The method of claim 1 , wherein the recombination system comprises a 3′-5′ exonuclease, a DNA polymerase, and a DNA ligase.

6. The method of claim 1 , wherein the CRISPR enzyme is coupled to a sgRNA.

7. The method of claim 1 , wherein the heterologous nucleic acid comprises an open reading frame that encodes a bioluminescent protein, a fluorescent protein, a chemiluminescent protein, or any combination thereof.

8. The method of claim 7 , wherein the open reading frame of the heterologous nucleic acid is operably linked to an expression control sequence that is capable of directing expression of the bioluminescent protein, the fluorescent protein, the chemiluminescent protein, or any combination thereof.

9. The method of claim 8 , wherein the expression control sequence is an inducible promoter or a constitutive promoter.

10. The method of claim 7 , wherein the chemiluminescent protein is β-galactosidase, horseradish peroxidase (HRP), or alkaline phosphatase.

11. The method of claim 7 , wherein the bioluminescent protein is Aequorin, firefly luciferase, Renilla luciferase, red luciferase, luxAB, or nanoluciferase.

12. The method of claim 7 , wherein the fluorescent protein is TagBFP, Azurite, EBFP2, mKalama1, Sirius, Sapphire, T-Sapphire, ECFP, Cerulean, SCFP3A, mTurquoise, monomeric Midoriishi-Cyan, TagCFP, mTFP1, EGFP, Emerald, Superfolder GFP, Monomeric Azami Green, TagGFP2, mUKG, mWasabi, EYFP, Citrine, Venus, SYFP2, TagYFP, Monomeric Kusabira-Orange, mKOK, mKO2, mOrange, mOrange2, mRaspberry, mCherry, dsRed, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, mRuby, mPlum, HcRed-Tandem, mKate2, mNeptune, NirFP, TagRFP657, IFP1.4, iRFP, mKeima Red, LSS-mKate1, LSS-mKate2, PA-GFP, PAmCherry1, PATagRFP, Kaede (green), Kaede (red), KikGR1 (green), KikGR1 (red), PS-CFP2, PS-CFP2, mEos2 (green), mEos2 (red), PSmOrange, or Dronpa.

Assignees

• Charles Stark Draper Laboratory Inc

Inventors

• Mcbrine Connor
• Holder Jason

Classifications

• C12N15/1072 Primary

  Differential gene expression library synthesis, e.g. subtracted libraries, differential screening · CPC title

• C12N2795/10243

  viral genome or elements thereof as genetic vector · CPC title

• C12N7/00 Primary

  Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) · CPC title

• C12N15/902

  using homologous recombination · CPC title

• C12N15/113

  Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)} · CPC title