Improving sequence-specific antimicrobials by blocking DNA repair

a technology of endonuclease and antimicrobial agent, which is applied in the direction of antibacterial agents, viruses/bacteriophages, peptide/protein ingredients, etc., can solve the problems of insufficient use of nucleases and bacteria, and bacteria will be susceptible to antibiotics without dna repair, so as to improve the ability of endonuclease to kill bacteria.

Inactive Publication Date: 2018-07-19
INST PASTEUR +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0006]Surprisingly, the inventors of the present invention found that combining the action of an endonuclease with the action of some protein...

Problems solved by technology

When a double strand beak is introduced at a given position in all copies of an antibiotic resistance plasmid simultaneously, the ba...

Method used

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  • Improving sequence-specific antimicrobials by blocking DNA repair
  • Improving sequence-specific antimicrobials by blocking DNA repair
  • Improving sequence-specific antimicrobials by blocking DNA repair

Examples

Experimental program
Comparison scheme
Effect test

example 1

Effect of Double Strand Breaks Introduced by Cas9 on Cell Death and Conditions for Survival to Such DNA Damage

[0130]Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated (Cas) genes are the adaptive immune system of bacteria and archaea [1]. The RNA-guided Cas9 nuclease from Streptococcus pyogenes has emerged as a useful and versatile tool [2]. The ease with which it can be reprogrammed has in particular been driving its adoption for genome editing applications. Cas9 is guided by a small CRISPR RNA (crRNA) that is processed from the initial transcript of the CRISPR locus by Cas9 together with a trans-activating CRISPR RNA (tracrRNA) and the host RNAselII [3]. Both the tracrRNA and the processed crRNA remain bound to Cas9 and act as a complex to direct interference against target DNA molecules[4]. Alternatively, the crRNA and tracrRNA can be fused forming a chimeric single guide RNA (sgRNA) [4]. Cas9 scans DNA looking for a short sequence motif know...

example 2

Bacterial Strains and Media

[0134]E. coli strains were grown in Luria-Bertani (LB) broth (10 g Tryptone, 5 g Yeast Extract, 10 g NaCl, add ddH2O to 1000 ml, PH7.5, autoclaved). 1.5% LB Agar was used as solid medium. Different antibiotics (20 ug / ml chloramphenicol, 100 ug / ml carbenicillin, 50 ug / ml kanamycin) were used as needed. Plates containing IPTG (100 uM) and X-gal (40 ug / ml) were used for blue / white screening. Escherichia coli strain MG1656 (a ΔlacI-lacZ derivative of MG1655) was used as a cloning strain for plasmid pCas9::lacZ2 (see below). E. coli strains N4278 (MG1655 recB268::Tn10)29, MG1655 RecA::Tn10 and JJC443 (lexAind3 MalF::Tn10)30 are gifts from the Mazel lab.

example 3

Plasmid Cloning

[0135]pCRRNA was assembled by amplification of pCRISPR using primer B299 / LC34 and of the tracrRNA fragment from pCas9 using primers LC35 / LC36, followed by Gibson assembly [31]. Novel spacers were cloned into pCRRNA or pCas9 plasmids as previously described [7]. The vector was digested with BsaI, followed by ligation of annealed oligonucleotides designed as follows: 5′-aaac+(target sequence)+g-3′ and 5′-aaaac+(reverse complement of the target sequence)-3′. A list of all spacers tested in this study is provided in (Table 2 in the present application was indicated with the number 4 in the text of the priority application corresponding to the table 2 of the priority application).

[0136]The pLCX plasmid was assembled from the pCRISPR backbone amplified using primers LC41 / LC42 and two lacZ fragments amplified from MG1655 genomic DNA using primers LC38 / LC39 and LC37 / LC40. The pZA31-sulA-GFP plasmid was assembled from pZA31-Luc [32] linearized with primers LC192 / LC193, the sul...

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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.

Description

FIELD OF THE INVENTION[0001]The invention relates to endonuclease-based antimicrobials that generate 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. The invention also relates to a vector encoding such endonuclease and a protein blocking DNA repair, a pharmaceutical composition and a product comprising said vector for use in the treatment of diseases dues to a bacterium infectionBACKGROUND OF THE INVENTION[0002]Cas proteins such as Cas9, of CRISPR-Cas systems, are members of the programmable nucleases, that have emerged as popular tools to introduce mutations in eukaryotic genomes as also are Zinc Finger Nucleases (ZFN) or Transcription Activator-Like Effector Nucleases (TALEN). Double strand breaks introduced in genomes by these nucleases can be repaired either through Homology Directed Repair (HDR) or through Non-Homologous End Joining (NH...

Claims

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Application Information

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IPC IPC(8): A61K38/46C12N15/63A61P31/04C12N15/10
CPCA61K38/465C12N15/63A61P31/04C12N15/1024C12N2795/10122C12N2310/20C12N1/20C12N9/22C12N15/10C12N15/70Y02A50/30
Inventor BIKARD, DAVIDCUI, LUNDUPORTET, XAVIERFERNANDEZ RODRIGUEZ, JESUS
Owner INST PASTEUR
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