Targeted elimination of bacterial genes

Abstract

The invention related to a kit or a system comprising two elements or components. The first component (i) is a selective component comprising a nucleic acid sequence comprising at least one proto-spacer. The second component (ii) comprises at least one sensitizing component comprising at least one cas gene and at least one CRISPR array. It should be noted that at least one spacer of the CRISPR targets a proto-spacer comprised within a pathogenic gene of a bacterium so as to specifically inactivate said pathogenic gene in said bacterium and wherein at least one spacer of said CRISPR targets a proto-spacer comprised within said selective component of (i) so as to specifically inactivate said selective component. The invention further provides method using the components or kits of the invention for interference with a horizontal transfer of a pathogenic gene between bacteria and for preventing a pathologic condition in a mammalian subject caused by a bacterial infection.

1 . A kit comprising:
 (i) at least one selective component comprising a nucleic acid sequence comprising at least one proto-spacer; and   (ii) at least one sensitizing component comprising at least one cas gene and at least one clustered, regularly interspaced short palindromic repeat (CRISPR) array, wherein at least one spacer of said CRISPR targets a proto-spacer comprised within at least one pathogenic gene or unit of genetic material, of a bacterium so as to specifically inactivate said pathogenic gene or genetic material in said bacterium and wherein at least one spacer of said CRISPR targets a proto-spacer comprised within said selective component of (i), so as to specifically inactivate said selective component.   
     
     
         2 . The kit according to  claim 1 , wherein said selective component comprises at least one genetic element, said genetic element is a lytic bacteriophage or a vector that encode a toxic element or protein that kill bacterial cells or disrupt and/or inhibit bacterial growth. 
     
     
         3 . The kit according to  claim 2 , wherein said lytic bacteriophage or a vector that encode a toxic element or protein that kill bacterial cells or disrupt and/or inhibit bacterial growth, is at least one genetically modified bacteriophage comprising at least one proto-spacer having an identity of at least 70% to at least one nucleic acid sequence comprised within said bacterial pathogenic gene. 
     
     
         4 . The kit according to  claim 1 , wherein said sensitizing component comprises at least one vector comprising a nucleic acid sequence encoding at least one cas protein, said vector further comprises a nucleic acid sequence of at least one of said CRISPR array, optionally, wherein said vector is at least one of genetically modified bacteriophage, plasmid, phagemide, virus and integratable DNA fragment, comprising at least one CRISPR spacer that targets at least one nucleic acid sequence comprised within said lytic bacteriophage or a vector that encode a toxic element or protein that kill bacterial cells or disrupt and/or inhibit bacterial growth, and at least one CRISPR spacer that targets a nucleic acid sequence comprised within said at least one pathogenic gene or unit of genetic material, thereby targeting and inactivating both, said lytic phage and said pathogenic gene or unit of genetic material. 
     
     
         5 . The kit according to  claim 1 , wherein:
 (a) said at least one bacterial pathogenic gene is at least one bacterial endogenous gene; or   (b) wherein said at least one bacterial pathogenic gene is at least one epichromosomal gene.   
     
     
         6 . The kit according to  claim 5 , wherein:
 (a) at least one of said pathogenic gene is an antibiotic resistance gene:   (b) at least one of said pathogenic gene is a gene encoding at least one of a virulence factor and at least one toxin.   
     
     
         7 . The kit according to  claim 5 , wherein said at least one antibiotic resistance gene encodes a resistance factor selected from the group consisting of CTX-M-15, New Delhi metallo-β-lactamase (NDM)-1, 2, 5, 6, an extended-spectrum beta-lactamase resistance factor (ESBL factor), beta lactamase, and tetracycline A (tetA), optionally, wherein said at least one CRISPR spacer comprises a nucleic acid sequence that targets at least one of: at least one proto-spacer of CTX-M-15, at least one proto-spacer of NDM-1, 2, 5, 6, at least one proto-spacer of ESBL factor, at least one proto-spacer of beta lactamase, at least one proto-spacer of tetA and at least one at least one proto-spacer of said lytic bacteriophage. 
     
     
         8 . The kit according to  claim 4 , wherein said at least one CRISPR spacer targets a nucleic acid sequence comprised within an essential gene of said lytic bacteriophage. 
     
     
         9 . The kit according to  claim 3 , wherein said lytic bacteriophage is at least one of T7like-virus and T4like-virus. 
     
     
         10 . The kit according to  claim 1 , wherein said at least one cas gene is at least one cas gene of at least one of type I, type II and type III CRISPR systems, optionally, said at least one cas gene is at least one cas gene of type I-E CRISPR system. 
     
     
         11 . The kit according to  claim 1 , wherein said bacterium is at least one bacterium of any strain of at least one of  E. coli, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pyogenes, Clostidium difficile, Enterococcus faecium, Klebsiella pneumonia, Acinetobacter baumanni  and  Enterobacter  species, optionally, wherein said bacteria are at least one  E. coli  strain selected from the group consisting of K-12, O157:H7, enteroaggregative (EAEC), enterohemorrhagic (EHEC), enteroinvasive (EIEC), enteropathogenic (EPEC), enterotoxigenic (ETEC) and diffuse adherent (DAEC)  E. coli.    
     
     
         12 . A method of interfering with a horizontal transfer of a genetic element comprising at least one pathogenic gene or unit of genetic material between bacteria, the method comprises the steps of: contacting at least one of a tissue, a surface, a substance and an article containing bacteria harboring said pathogenic gene or unit of genetic material with at least one of:
 (i) at least one selective component comprising a nucleic acid sequence comprising at least one proto-spacer;   (ii) at least one sensitizing component comprising at least one cas gene and at least one CRISPR array, wherein at least one spacer of said CRISPR, targets a proto-spacer comprised within at least one pathogenic gene or unit of genetic material, of a bacterium so as to specifically inactivate said pathogenic gene in said bacterium and wherein at least one spacer of said CRISPR targets a proto-spacer comprised within said selective component of (i) so as to specifically inactivate said selective component, and   (iii) at least one kit comprising (i) and (ii), thereby inactivating said pathogenic gene or unit of genetic material and interfering with horizontal transfer thereof.   
     
     
         13 . The method according to  claim 12 , wherein said selective component comprises at least one genetic element, said genetic element is a lytic bacteriophage or a vector that encode a toxic element or protein that kill bacterial cells or disrupt and/or inhibit bacterial growth. 
     
     
         14 . A method of preventing a pathologic condition in a mammalian subject caused by or associated with a bacterial infection of bacteria containing a pathogenic gene or unit of genetic material, the method comprising contacting at least one of a tissue, a surface, a substance and an article, optionally, in the vicinity of said subject with at least one of:
 (i) at least one selective component comprising a nucleic acid sequence comprising at least one proto-spacer;   (ii) at least one sensitizing component comprising at least one cas gene and at least one clustered, regularly interspaced short palindromic repeat (CRISPR) array, wherein at least one spacer of said CRISPR targets a proto-spacer comprised within at least one pathogenic gene or unit of genetic material of a bacterium so as to specifically inactivate said pathogenic gene in said bacterium and wherein at least one spacer of said CRISPR targets a proto-spacer comprised within said selective component of (i) so as to specifically inactivate said selective component, and   (iii) at least one kit comprising (i) and (ii), thereby targeting and inactivating said pathogenic gene and preventing said pathologic condition.   
     
     
         15 . The method according to  claim 14 , comprising contacting said at least one of tissue, surface, substance and article with said sensitizing component and subsequently contacting said solid surface with said selective component. 
     
     
         16 . The method according to  claim 14 , wherein at least one of:
 (a) said selective component comprises at least one genetic element, said genetic element is a lytic bacteriophage or a vector that encode a toxic element or protein that kill bacterial cells or disrupt and/or inhibit bacterial growth; and   (b) said sensitizing component comprises at least one vector comprising a nucleic acid sequence encoding at least one cas protein, said vector further comprises a nucleic acid sequence of at least one of said CRISPR array, optionally, wherein said vector is at least one of genetically modified bacteriophage, plasmid, phagemide, virus and integratable DNA fragment, comprising at least one CRISPR spacer that targets at least one nucleic acid sequence comprised within said lytic bacteriophage or a vector that encode a toxic element or protein that kill bacterial cells or disrupt and/or inhibit bacterial growth, and at least one CRISPR spacer that targets a nucleic acid sequence comprised within said at least one pathogenic gene or unit of genetic material, thereby targeting and inactivating both, said lytic phage and said pathogenic gene or unit of genetic material.   
     
     
         17 . A method for sensitizing bacterial population or increasing the sensitivity of said population to at least one antibiotic compound, and/or for preventing or reducing resistance of bacteria or bacterial population/s to at least one antibiotic compound, the method comprising applying on said bacterial population or on at least one of a tissue, a surface, a substance and an article containing said bacterial population with the kit of  claim 1 , the kit comprising:
 (i) at least one selective component comprising a nucleic acid sequence comprising at least one proto-spacer;   (ii) at least one sensitizing component comprising at least one cas gene and at least one CRISPR array, wherein at least one spacer of said CRISPR targets a proto-spacer comprised within at least one pathogenic gene or a unit of genetic material of a bacterium, that provides resistance of said bacterium to said antibiotic compound, so as to specifically inactivate said pathogenic gene or unit of genetic material in said bacterium, and wherein at least one spacer of said CRISPR targets a proto-spacer comprised within said selective component of (i) so as to specifically inactivate said selective component, and   (iii) at least one kit comprising (i) and (ii), thereby inactivating said pathogenic gene or unit of genetic material and sensitizing said bacterial population to said at least one antibiotic compound.   
     
     
         18 . The method according to  claim 17 , wherein at least one of:
 (a) said selective component comprises at least one genetic element, said genetic element is a lytic bacteriophage or a vector that encode a toxic element or protein that kill bacterial cells or disrupt and/or inhibit bacterial growth; and   (b) said sensitizing component comprises at least one vector comprising a nucleic acid sequence encoding at least one cas protein, said vector further comprises a nucleic acid sequence of at least one of said CRISPR array, optionally, wherein said vector is at least one of genetically modified bacteriophage, plasmid, phagemide, virus and integratable DNA fragment, comprising at least one CRISPR spacer that targets at least one nucleic acid sequence comprised within said lytic bacteriophage or a vector that encode a toxic element or protein that kill bacterial cells or disrupt and/or inhibit bacterial growth, and at least one CRISPR spacer that targets a nucleic acid sequence comprised within said at least one pathogenic gene or unit of genetic material, thereby targeting and inactivating both, said lytic phage and said pathogenic gene or unit of genetic material.   
     
     
         19 . The method of  claim 17 , for preventing or reducing resistance of bacteria or bacterial population/s to at least one antibiotic compound, the method comprising the steps of: contacting said bacteria or bacterial population/s, or at least one of a tissue, a surface, a substance and an article containing bacteria harboring at least one pathogenic gene or a unit of genetic material that provide resistance to said at least one genetic compound with at least one of:
 (i) at least one selective component comprising a nucleic acid sequence comprising at least one proto-spacer;   (ii) at least one sensitizing component comprising at least one cas gene and at least one CRISPR array, wherein at least one spacer of said CRISPR targets a proto-spacer comprised within said at least one pathogenic gene or unit of genetic material of a bacterium so as to specifically inactivate said pathogenic gene in said bacterium and wherein at least one spacer of said CRISPR targets a proto-spacer comprised within said selective component of (i) so as to specifically inactivate said selective component, and   (iii) at least one kit comprising (i) and (ii), thereby inactivating said pathogenic gene or unit of genetic material and reducing resistance of said bacteria or bacterial population to said at least one antibiotic compound.   
     
     
         20 . The method according to  claim 19 , wherein at least one of:
 (a) said selective component comprises at least one genetic element, said genetic element is a lytic bacteriophage or a vector that encode a toxic element or protein that kill bacterial cells or disrupt and/or inhibit bacterial growth; and   (b) said sensitizing component comprises at least one vector comprising a nucleic acid sequence encoding at least one cas protein, said vector further comprises a nucleic acid sequence of at least one of said CRISPR array, optionally, wherein said vector is at least one of genetically modified bacteriophage, plasmid, phagemide, virus and integratable DNA fragment, comprising at least one CRISPR spacer that targets at least one nucleic acid sequence comprised within said lytic bacteriophage or a vector that encode a toxic element or protein that kill bacterial cells or disrupt and/or inhibit bacterial growth, and at least one CRISPR spacer that targets a nucleic acid sequence comprised within said at least one pathogenic gene or unit of genetic material, thereby targeting and inactivating both, said lytic phage and said pathogenic gene or unit of genetic material.