Abolishment of antibiotic resistance in Escherichia Coli using a conjugative CRISPR-Cas9 plasmid
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2020Copyright
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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.
...




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