Bacteriophage recombination followed by blockage of non-recombinant bacteriophage replication

Patent metadata

Field	Value
Publication number	US-2018223291-A1
Application number	US-201815893104-A
Country	US
Kind code	A1
Filing date	Feb 9, 2018
Priority date	Feb 9, 2017
Publication date	Aug 9, 2018
Grant date	—

Abstract

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

First claim

1 . A method for making a recombinant bacteriophage DNA genome in a first bacterial host cell comprising
(a) contacting a first bacteriophage DNA genome with a first sgRNA-CRISPR enzyme complex and a second sgRNA-CRISPR enzyme complex in vivo under conditions where
(i) the first sgRNA-CRISPR enzyme complex cleaves a first protospacer sequence within the first bacteriophage DNA genome; and
(ii) the second sgRNA-CRISPR enzyme complex cleaves a second protospacer sequence within the first bacteriophage DNA genome to produce a cleaved first bacteriophage DNA genome; and
(b) recombining in vivo the cleaved first bacteriophage DNA genome with a heterologous nucleic acid sequence in the presence of a recombination system under conditions to produce the recombinant bacteriophage DNA genome, wherein the first bacterial host cell is infected with the first bacteriophage DNA genome, and wherein the heterologous nucleic acid sequence comprises an open reading frame that encodes a bioluminescent protein, a fluorescent protein, a chemiluminescent protein, or any combination thereof.

2 . The method of claim 1 , wherein the first bacterial host cell may be a non-natural bacterial host cell or a natural bacterial host cell for the recombinant bacteriophage DNA genome.

3 . The method of claim 1 , wherein the first bacteriophage DNA genome corresponds to a bacteriophage family or order selected from the group consisting of Myoviridae, Siphoviridae, Podoviridae, Lipothrixviridae, Rudiviridae, Ampullaviridae, Bucaudaviridae, Clavaviridae, Corticoviridae, Cystoviridae, Fuselloviridae, Globuloviriade, Guttaviridae, Inoviridae, Leviviridae, Mircoviridae, Plasmaviridae, and Tectiviridae.

4 . The method of claim 1 , wherein the first bacteriophage DNA genome corresponds to T3, T7, M6, K11, F92, K1-5, or K1F.

5 . The method of claim 1 , wherein the cleaved first bacteriophage DNA genome comprises a first cleaved bacteriophage genomic fragment and a second cleaved bacteriophage genomic fragment.

6 . The method of claim 5 , wherein the heterologous nucleic acid sequence comprises a 5′ flanking region that is homologous to the 3′ end of the first cleaved bacteriophage genomic fragment, and a 3′ flanking region that is homologous to the 5′ end of the second cleaved bacteriophage genomic fragment.

7 . The method of claim 1 , wherein the recombination system is endogenous to the first bacterial host cell.

8 . The method of claim 1 , wherein the recombination system is a non-endogenous recombination system that is induced in the first bacterial host cell.

9 . The method of claim 8 , wherein the non-endogenous recombination system comprises lambda Red proteins Gam, Exo, and Beta operably linked to an inducible promoter.

10 . The method of claim 8 , wherein the non-endogenous recombination system comprises Exo, RecA, and Gam proteins operably linked to an inducible promoter.

11 . The method of claim 1 , wherein the first bacterial host cell comprises a first non-endogenous CRISPR expression vector comprising a nucleic acid sequence that encodes a first sgRNA, a second sgRNA, and a first CRISPR enzyme.

12 . The method of claim 11 , wherein the first sgRNA and the second sgRNA are operably linked to a constitutive promoter.

13 . The method of claim 11 , wherein the first CRISPR enzyme is a Cas protein selected from the group consisting of Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cash, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, and Csf4, and optionally wherein the first CRISPR enzyme is operably linked to an inducible promoter.

14 . The method of claim 1 , wherein the bioluminescent protein is Aequorin, firefly luciferase, Renilla luciferase, red luciferase, luxAB, or nanoluciferase.

15 . The method of claim 1 , wherein the chemiluminescent protein is (3-galactosidase, horseradish peroxidase (HRP), or alkaline phosphatase.

16 . The method of claim 1 , wherein the fluorescent protein is TagBFP, Azurite, EBFP2, mKalamal, 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-mKatel, LSS-mKate2, PA-GFP, PAmCherryl, PATagRFP, Kaede (green), Kaede (red), KikGR1 (green), KikGR1 (red), PS-CFP2, PS-CFP2, mEos2 (green), mEos2 (red), PSmOrange, or Dronpa.

17 . The method of claim 1 , 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.

18 . The method of claim 17 , wherein the expression control sequence is an inducible promoter or a constitutive promoter.

19 . The method of claim 1 , wherein the heterologous nucleic acid is about 100-500 base pairs in length or about 500-1500 base pairs in length.

20 . The method of claim 11 , further comprising propagating the recombinant bacteriophage DNA genome in the first bacterial host cell.

21 . The method of claim 1 , further comprising propagating the recombinant bacteriophage DNA genome in a second bacterial host cell.

22 . The method of claim 21 , wherein the second bacterial host cell comprises a second non-endogenous CRISPR expression vector comprising a nucleic acid sequence that encodes a second CRISPR enzyme, a third sgRNA, and a fourth sgRNA, wherein the third sgRNA and the fourth sgRNA are operably linked to a constitutive promoter, and wherein the second CRISPR enzyme is operably linked to an inducible promoter.

23 . The method of claim 22 , wherein the third sgRNA binds to the second CRISPR enzyme to form a third sgRNA-CRISPR enzyme complex, and the fourth sgRNA binds to the second CRISPR enzyme to form a fourth sgRNA-CRISPR enzyme complex.

24 . The method of claim 23 , wherein the third sgRNA-CRISPR enzyme complex and the fourth sgRNA-CRISPR enzyme complex (a) do not cleave the recombinant bacteriophage DNA genome and (b) cleave the first bacteriophage DNA genome.

25 . The method of claim 22 , wherein the second CRISPR enzyme is Cas9.

26 . The method of claim 21 , wherein the second bacterial host cell comprises a non-endogenous Cascade complex expression vector comprising a nucleic acid sequence that encodes a casABCDE operon, and Cas3 nuclease.

27 . The method of claim 26 , wherein the casABCDE operon and Cas3 nuclease are operably linked to an inducible promoter or a constitutive promoter.

28 . The method of claim 26 , wherein the second bacterial host cell comprises a non-endogenous CRISPR RNA (crRNA) spacer array comprising one or more spacers that (a) induce cleavage in the first bacteriophage DNA genome and (b) do not induce cleavage in the recombinant bacteriophage DNA genome.

Assignees

• Charles Stark Draper Laboratory Inc

Inventors

• Holder Jason
• Mcbrine Connor
• Rogers Miles
• Billings Nicole
• Gruska Sarah
• Tilley Lucas

Classifications

• C07K14/005

  from viruses · CPC title

• C12N2795/10221

  Viruses as such, e.g. new isolates, mutants or their genomic sequences · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title

• C12N15/11

  DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity} · CPC title

• C12N15/70 Primary

  Vectors or expression systems specially adapted for E. coli · CPC title