Improving sequence-specific antimicrobials by blocking dna repair

Abstract

The invention relates to the improvement of endonuclease-based antimicrobials by blocking DNA repair of double-strand break(s) (DSB(s)) in prokaryotic cells. In this respect, the invention especially concerns a method involving blocking DNA repair after a nucleic acid has been submitted to DSB, in particular by a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated programmable double-strand endonuclease. The invention particularly relates to the use of an exogenous molecule that inhibits DNA repair, preferably a protein that binds to the ends of the double-stranded break to block DSB repair. The invention also relates to vectors, particularly phagemids and plasmids, comprising nucleic acids encoding nucleases and Gam proteins, and a pharmaceutical composition and a product containing these vectors and their application.

1 - 27 . (canceled) 
     
     
         28 . A method for killing a bacterium comprising contacting the bacterium with at least one recombinant phagemid(s) or plasmid(s);
 wherein the recombinant phagemid(s) or plasmid(s) encodes an endonuclease that creates a double-stranded break (DSB) in the chromosomal or extrachromosomal DNA of the bacterium, and   an exogenous protein that inhibits DSB repair.   
     
     
         29 . The method of  claim 28 , wherein the exogenous protein is encoded by the same vector as the endonuclease or by a separate vector. 
     
     
         30 . The method of  claim 28 , wherein the protein is synthetized before contacting with the bacterium. 
     
     
         31 . The method of  claim 28 , wherein the endonuclease is selected from a meganuclease or an artificial endonuclease. 
     
     
         32 . The method of  claim 28 , wherein the endonuclease specifically cleaves the chromosomal or extrachromosomal DNA of the bacterium at less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different sites. 
     
     
         33 . The method of  claim 28 , wherein the at least one recombinant phagemid(s) or plasmid(s) encodes a Cas9 nuclease, a guide RNA, and an exogenous protein that inhibits DNA repair selected from the group consisting of a Mu phage Gam protein, a lambda phage Gam protein, and a phage T7 gp5.9 protein. 
     
     
         34 . The method of  claim 28 , wherein the at least one recombinant phagemid(s) is selected from the group consisting of M13, lambda, p22, T7, Mu, T4 phage, PBSX, P1Puna-like, P2, 13, Bcep 1, Bcep 43, Bcep 78, T5 phage, phi, C2, L5, HK97, N15, T3 phage, P37, MS2, Qβ, or Phi X 174, T2 phage, T12 phage, R17 phage, M13 phage, G4 phage, Enterobacteria phage P2, P4 phage, N4 phage,  Pseudomonas  phage ϕ6, ϕ29 phage and 186 phage. 
     
     
         35 . The method of  claim 28 , wherein the bacterium comprises a recBCD homologous repair pathway or addAB system. 
     
     
         36 . The method of  claim 28 , wherein the bacterium is selected from the group consisting of  Enterobacter, Streptococci, Staphylococci, Enterococci, Salmonella, Pseudomonas , and  Mycobacterium.    
     
     
         37 . The method of  claim 28  wherein the recombinant phagemid(s) or plasmid(s) encode(s) an endonuclease that creates a double-stranded break (DSB) in an antibiotic resistance gene encoded by the bacterium 
     
     
         38 . λ phagemid or plasmid vector encoding an endonuclease and an exogenous protein inhibiting DSB repair. 
     
     
         39 . The phagemid or plasmid vector of  claim 38  wherein the recombinant phagemid(s) is selected from the group consisting of M13, lambda, p22, T7, Mu, T4 phage, PBSX, P1Puna-like, P2, 13, Bcep 1, Bcep 43, Bcep 78, T5 phage, phi, C2, L5, HK97, N15, T3 phage, P37, MS2, Qβ, or Phi X 174, T2 phage, T12 phage, R17 phage, M13 phage, G4 phage, Enterobacteria phage P2, P4 phage, N4 phage,  Pseudomonas  phage ϕ6, ϕ29 phage and 186 phage. 
     
     
         40 . The phagemid or plasmid vector of  claim 38 , wherein the phagemid vector is a P1 bacteriophage. 
     
     
         41 . The phagemid or plasmid vector of  claim 38 , wherein the phagemid vector is a λ bacteriophage. 
     
     
         42 . A pharmaceutical composition comprising a phagemid or plasmid vector encoding an endonuclease, and an exogenous protein inhibiting DSB repair or a vector encoding an exogenous protein inhibiting DSB repair, and a pharmaceutically acceptable vehicle. 
     
     
         43 . The pharmaceutical composition of  claim 42  further comprising an antibiotic. 
     
     
         44 . The pharmaceutical composition of  claim 42  containing a phagemid or plasmid vector encoding an endonuclease and a vector encoding an exogenous protein inhibiting DSB repair. 
     
     
         45 . The pharmaceutical composition of  claim 42 , wherein said exogenous protein is encoded by the same vector as the endonuclease.