Eradication To Bacterial Antimicrobial Resistance With Temperate Phage-delivered CRISPR-Cas9 System

Backgroud:In recent years,pan-drug resistant and multi-drug resistant bacteria emerged with increasing rate.Bacterial resistance against first-line antibiotics became more and more prevelant.Especially since the emergence of super-resistant microbes,clinical anti-infection treatment faced the situation of no cure.Increasing prevalence of bacterial drug resistance has become a global health problem.Responsible resistance genes tend to locate in bacterial plasmids,with high horizontal transfer ability.At present,there were seldom effective solutions for treatment of pan-and super-resistant bacteria,nor for the control of spreading bacterial drug resistance.New technology and strategies are in urgent need in treating and controling drug-resistant bacteria.Aim:Bacterial resistance is mainly mediated and transmitted by resistance plasmids.Using prokaryotic CRISPR-Cas9 gene editing technology to remove bacterial resistance plasmids can restore the antibacterial efficacy of antibiotics and block the spread of bacterial resistance.With more efficient prokaryotic vector techniques,the CRISPR-Cas9 system eradicating bacterial-resistant plasmid can be delivered into bacterial cells more efficiently,which will greatly enhance the practical potential of this system in the field of defending resistant bacteria.This study will establish a CRISPR-Cas9 technology that efficiently removes bacterial resistance.On this basis,temperate phage will be utilized as efficient delivery vector of CRISPR-Cas9 sysstem.A novel temperate phage packaging CRISPR-Cas9 technology is designed,to achieve"one-step"packaging of the CRISPR system.We will explore and establish the technical protocols for phage package,from the separation of new phage to the completion of packaging.The phage-delivered CRISPR-Cas9 system would be examined for its sensitization efficiency and bacticidal efficiency to resistant bacteria combined with antibiotics in a series of in vitro and in vivo experiments.Through long-term monitoring of the bactericidal effect,it would be revealed whether the modified phage treatment can avoid bacterial resistance mutations and whether it has a longer inhibiton effect to bacterial growth than lytic phage therapy.Methods:A CRISPR-Cas9 spacer library against the super-resistant gene bla_NDM-1 was constructed.CRISPR-Cas9 system plasmids were constructed to target NDM-1plasmids.Then the drug-resistant E.coli strain models carrying NDM-1 plasmid were constructed,and the efficiency of CRISPR-Cas9 system to remove NDM-1 plasmid was analyzed by quantitative PCR and fluorescent signal detection.The drug sensitivity test was used to test the bacterial resistance phenotype after resistance plasmid clearance.These assays were arranged to verify the resistant plasmid eradication effect of CRISPR-Cas9 system.A novel recombinant phage screening technique,other than resistance marker screening,was designed to establish a"one-step"phage packaging technology for CRISPR-Cas9.A newly isolated E.coli temperate phage was engineered using this technique to deliver the CRISPR-Cas9 system targeting bla_NDM-1.E.coli host cells carrying the target plasmid was infected with the modified phage to analyse the plasmid eradication effect of phage-delivered CRISPR.The clearance effect to the target plasmid was detected by PCR;the target plasmid removal efficiency was quantitatively analyzed by qPCR;and the sensitization rate of the resistant bacteria was caculated by the colony counting method.The potential effect of the phage delivered CRISPR-Cas9 system to block the spread of bacterial resistance was evaluated by the plasmid transformation and conjugation assays.A combinaroty strategy was established that killing drug-resistant bacteria by modified phage and sensitized antibiotics.Effectiveness of this strategy was tested for resistance plasmid clearance and drug-resistant bacteria killing in vivo and in vitro conditions including biofilm,mouse skin infection model,and mouse intestinal infection model.The bactericidal efficacy of the combined anti-drug resistant strategy was monitored continuously for at least one week to find if resistant bacterial mutant were observed,and compared with the appearance of induced resistance mutant in the lytic bacteriophage bactericidal strategy.Whole genomic sequencing was used to analyze the MBEChanism by which bacteria produce induced resistance mutations.Result:（1）Library of specific spacer sequence targeting bla_NDM-1 gene was established,including sequences of 20 specific spacers.Specific CRISPR-Cas9 targeting bla_NDM-1gene was constructed in plasmid vectors.（2）Removal of NDM-1-encoding plasmid in E.coli cells by CRISPR technology turned the bacteria a bla_NDM-1 negative strain,and restored the sensitivity of imipenem and other beta-lactam antibiotics.（3）NDM-1 plasmids carried by E.coli cells were eradicated efficiently after transformation of specific CRISPR-Cas9 plasmid.Both natural large NDM-1 plasmid and restructured high copy NDM-1 plasmid can be removed by more than 99%clearance rate in 12 h.（4）New technology for CRISPR-Cas9 packaging with phage vectors was designed.The complete technical process from new E.coli temperate phage isolation and sequencing to the integration of CRISPR-Cas9 cassette was established for the first time.（5）Clearance efficiency of target plasmid by phage-delivered CRISPR-Cas9system was evaluated in vitro,proved over 99%clearance rate in 8 hours.We designed the strategy of killing resitant bacteria using phage-delivered CRISPR and followed use of re-sensitized antibiotic.In vitro experiments showed over 10⁶ decrease to the counts of resistant E.coli cells using this strategy,which is more efficient than bacterial chromosome targeting strategies reported in former researches.（6）In vitro application of modified phage can reduce the accumulation of bacteria resistant plasmid in the environment,block the transfer of resistance plasmid by bacterial conjugation,and inhibit the spread of antibiotic resistance.It is expected to be developed as an active environmental application to facilitate the prevention and control of the spreading drug-resistant genes.（7）This research applied phage-delivered CRISPR technology in biofilm formated drug-resistant bacteria for the first time,and proved that this technology can remove resistant plasmids in bacteria harboured in biofilm and effectively reduce the overall resistance of biofilm.（8）Plasmid-targeting strategy was evaluated for the first time in in vivo assays for the first time.Phage-delivered CRISPR system can eradicate bacterial resistant plasmids in mice model of skin or intestinal infection.Combined use of antibiotics can kill drug-resistant bacteria,with a reduction of target drug-resistant E.coli cells by 10⁵¹0⁶times,which is more effective than that of reported chromosome-targeting strategy.（9）Through the long-term in vitro bactericidal effect monitoring,lytic bacteriophage vB₂53 was observed with new bacterial resistant in 24 h,while the new resistance of bacteria against the combined bactericidal strategy of modified phage with subsequent antibiotic was not observed within 192 hours.The strategy was further survellanced in in vivo conditions.Lytic phage was observed with new bacterial resistance within 2³ days in mouse skin or intestinal infection models,but no new mutational resistance were observed within 7 days using the combined strategy.（10）By whole genome sequencing analysis,it is revealed that bacterial resistance against lytic phage vB₂53was caused by base mutations at fepA gene.Mutational resistance against kanamycin treatment was caused by base mutations at fusA gene.The combinatory bactericidal strategy of phage-delivered CRISPR-Cas9 system with subsequent antibiotic was not observed with these bacterial mutations.Conclusions:This study proposes a strategy for the removal of bacterial resistance using the phage-ddelivered CRISPR-Cas9 system.A novel one-step phage packaging protocol for the CRISPR-Cas9 system was developed.The phage-delivered CRISPR-Cas9 system effectively eliminated bacterial resistance plasmids,destroyed the plasmid mediating bacterial resistance,blocked the spread of bacterial resistance,and reduced the level of resistance genes in the bacterial environment.It has the potential to advance as a new technology for the prevention and control of bacterial resistance.In the field of drug-resistant bacteria treatment,phage-delivered CRISPR-Cas9 eliminates bacterial resistance,then subsequent antibiotics revealed a potent bactericidal effect against resistant bacteria in vitro and in vivo.This combined strategy not only eradicate bacterial plasmid-mediated drug resistance and revive the antibiotic therapies,but also avoid the development of mutational resistance during treatment.It is expected to become a new method to effectively kill resistant bacteria with huge practical potential.