Phage engineering: protection by circularized intermediate

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.

1 . A kit comprising (a) one or more labeled vials containing a plurality of linear bacteriophage DNA genomes, (b) at least one CRISPR enzyme, (c) a recombination system, and (d) at least one exonuclease, and optionally instructions for use. 
     
     
         2 . The kit 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. 
     
     
         3 . The kit of  claim 1 , wherein the recombination system comprises a 5′-3′ exonuclease, a DNA polymerase, and a DNA ligase. 
     
     
         4 . The kit of  claim 1 , wherein the at least one 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. 
     
     
         5 . The kit of  claim 1 , wherein the recombination system comprises a 3′-5′ exonuclease, a DNA polymerase, and a DNA ligase. 
     
     
         6 . The kit of  claim 1 , wherein the plurality of linear bacteriophage DNA genomes correspond to a bacteriophage family or order selected from the group consisting of Myoviridae, Styloviridae, Siphoviridae, Pedoviridae, Tectiviridae, Leviviridae, Podoviridae, and Plasmaviridae. 
     
     
         7 . The kit of  claim 1 , wherein the plurality of linear bacteriophage DNA genomes are derived from one or more bacteriophage genuses (or genera) selected from the group consisting of T7-like phage, phiKMV-like phage, LUZ24-like phage, phiKZ-like phage, PB1-like phage, Felix-O1-like phage, T4-like phage, phi92-like phage, rV5-like phage, SP6-like phage, N4-like phage, phiEco32-like phage, T5-like phage, KP34-like phage, KP15-like phage, GAP227-like phage, AP22-like phage, phiFel-like phage, Sap6-like phage, Silvia-like phage, Kay-like phage, Twort-like phage, P68-like phage, and phiETA-like phage. 
     
     
         8 . The kit of  claim 4 , wherein the at least one CRISPR enzyme is coupled to a sgRNA. 
     
     
         9 . The kit of  claim 8 , wherein the sgRNA has the sequence of SEQ ID NO:2 or SEQ ID NO:3. 
     
     
         10 . The kit of  claim 1 , further comprising a heterologous nucleic acid. 
     
     
         11 . The kit of  claim 10 , 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. 
     
     
         12 . The kit of  claim 11 , wherein the bioluminescent protein is selected from the group consisting of Aequorin, firefly luciferase,  Renilla  luciferase, red luciferase, luxAB, and nanoluciferase. 
     
     
         13 . The kit of  claim 11 , wherein the fluorescent protein is selected from the group consisting of blue/UV fluorescent proteins (for example, TagBFP, Azurite, EBFP2, mKalama1, Sirius, Sapphire, and T-Sapphire), cyan fluorescent proteins (for example, ECFP, Cerulean, SCFP3A, mTurquoise, monomeric Midoriishi-Cyan, TagCFP, and mTFP1), green fluorescent proteins (for example, EGFP, Emerald, Superfolder GFP, Monomeric Azami Green, TagGFP2, mUKG, and mWasabi), yellow fluorescent proteins (for example, EYFP, Citrine, Venus, SYFP2, and TagYFP), orange fluorescent proteins (for example, Monomeric Kusabira-Orange, mKOK, mKO2, mOrange, and mOrange2), red fluorescent proteins (for example, mRaspberry, mCherry, dsRed, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, and mRuby), far-red fluorescent proteins (for example, mPlum, HcRed-Tandem, mKate2, mNeptune, and NirFP), near-IR fluorescent proteins (for example, TagRFP657, IFP1.4, and iRFP), long stokes-shift proteins (for example, mKeima Red, LSS-mKate1, and LSS-mKate2), photoactivatable fluorescent proteins (for example, PA-GFP, PAmCherryl, and PATagRFP), photoconvertible fluorescent proteins (for example, Kaede (green), Kaede (red), KikGR1 (green), KikGR1 (red), PS-CFP2, PS-CFP2, mEos2 (green), mEos2 (red), PSmOrange, and PSmOrange), fluorescein, rhodamine, and photoswitchable fluorescent proteins (for example, Dronpa). 
     
     
         14 . The kit of  claim 11 , wherein the chemiluminescent protein is selected from the group consisting of β-galactosidase, horseradish peroxidase (HRP), and alkaline phosphatase. 
     
     
         15 . The kit of  claim 11 , wherein the heterologous nucleic acid comprises an epitope that can be detected with an antibody or other binding molecule. 
     
     
         16 . The kit of  claim 11 , wherein the heterologous nucleic acid comprises a polypeptide tag sequence selected from among poly-histidine, FLAG, and Glutathione S-transferase (GST). 
     
     
         17 . The kit of  claim 11 , wherein the heterologous nucleic acid sequence comprises a biotin binding protein. 
     
     
         18 . The kit of  claim 17 , wherein the biotin binding protein is avidin, streptavidin, or neutrAvidin. 
     
     
         19 . The kit of  claim 17 , wherein the biotin binding protein is detected using a biotin molecule conjugated to an enzyme or an antibody. 
     
     
         20 . The kit of  claim 19 , wherein the enzyme is β-galactosidase, horseradish peroxidase (HRP), or alkaline phosphatase.