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Abolishment of antibiotic resistance in Escherichia Coli using a conjugative CRISPR-Cas9 plasmid

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Authors
Mikkola, Aapo
Date
2020
Discipline
Solu- ja molekyylibiologiaCell and molecular biology
Copyright
This publication is copyrighted. You may download, display and print it for Your own personal use. Commercial use is prohibited.

 
Kasvava antibioottiresistenssi on merkittävä ongelma, joka vaikuttaa globaalisti terveydenhoitoon, ruoantuotantoon ja taloudelliseen kasvuun. Beta-laktaamien antibioottiluokka kattaa noin kaksi kolmasosaa ihmisten käyttämistä antibiooteista. Laajan kirjon beta-laktamaasi (ESBL) -entsyymit mahdollistavat monipuolisen beta- laktaami-resistenssin useassa eri bakteerilajissa. Tässä tutkimuksessa kloonattiin konjugatiivinen CRISPR-Cas9-plasmidi, joka hyökkää pEC13 ESBL-plasmidia vastaan E. Coli-bakteerissa. Tutkimuksen CRISPR-Cas9-plasmidiin liitettiin oriT (origin of transfer) -sekvenssi, joka mahdollistaa tämän kokeellisen plasmidin konjugoimisen käyttäen toisen pLM2-plasmidin konjugaatiokoneistoa. CRISPR- Cas9-plasmidiin kohdentamiseksi pEC13-plasmidiin, siihen liitettiin myös IncFII crRNA-juoste. Tämän muokatun CRISPR-Cas9-plasmidin tulisi täten pystyä konjugoitumaan uuteen isäntäsoluun pLM2-plasmidin välityksellä, missä CRISPR- Cas9-plasmidin Cas9-endonukleaasi leikkaa pEC13-plasmidin DNA- kaksoisjuosteen sen IncFII-sekvenssin kohdalta. Leikatun pEC13-plasmidin ei tulisi enää selvitä isäntäsolussa, minkä tulisi johtaa isäntäsolun kuolemaan beta- laktaami-maljoilla. Tulokset osoittavat, että tutkimuksen CRISPR-Cas9 plasmidi konjugoituu uusiin isäntäsoluihin ja indusoitu CRISPR-Cas9-plasmidi vähentää beta-laktaamilla kasvavien isäntäbakteereiden määrää noin kahden kertaluokan verran. Kokonaisuutena, tätä tutkimusta voidaan pitää onnistuneena konseptin todistuksena, mikä näyttää, että kokeellinen CRISPR-Cas9-plasmidi on mahdollista konjugoida uuteen isäntäbakteeriin, missä sen CRISPR-Cas9-aktiivisuus huomattavasti heikentää isäntäbakteerien selviytymistä beta-laktaami-maljoilla. ...
 
Emerging antibiotic resistance is one of the major threats to modern healthcare as well as to global food security and economic development. Approximately two- thirds of antibiotics administered to humans are ß-lactams. The emergence of extended-spectrum ß-lactamases (ESBLs) in a variety of bacteria confers multi- resistance to ß-lactams. In this study, an ESBL-harbouring pEC13 plasmid was targeted with a CRISPR-Cas9 plasmid that is delivered to the target E. Coli cells via conjugation machinery of the conjugative plasmid pLM2. An anti-ESBL plasmid was cloned, which carries an origin of transfer (oriT) domain for the conjugation initiation along with a gene for CRISPR-Cas9. One specific guiding RNA targeting the IncFII replication initiator gene of the pEC13 plasmid was designed and annealed to the anti-ESBL plasmid. In practice, the introduction of a modified anti- ESBL plasmid through a conjugation channel into an ESBL-harbouring bacterium leads to the expression of guiding RNAs that direct the Cas9 endonuclease to cleave the ESBL-plasmid, thus compromising its maintenance in the host. The results show that the induced anti-ESBL plasmid reduces the colony forming unit count of the ESBL-harbouring host by approximately two orders of magnitude on ß-lactam plates. The same ß-lactam concentration was tested to be lethal without the pEC13 resistance plasmid. Consequently, this is a viable proof-of-principle study, which shows that an anti-ESBL CRISPR-Cas9 plasmid can be introduced into ESBL- bacteria via conjugation, and its directed endonuclease activity can substantially hinder the survival of those ESBL-bacteria in the presence of lethal ß-lactam concentration. ...
 
Keywords
ampicillin beta-lactam CRISPR-Cas9 conjugation plasmidit antibiootit antibioottiresistenssi kolibakteerit molekyyligenetiikka plasmids antibiotics antibiotic resistance Escherichia coli molecular genetics
URI

http://urn.fi/URN:NBN:fi:jyu-202103021816

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