%0 Journal Article %A Sünderhauf, David %A Pursey, Elizabeth %A Klümper, Uli %A Westra, Edze %A Gaze, William %A van Houte, Stineke %T AMR gene removal by conjugative delivery of CRISPR-Cas9 %D 2019 %J Access Microbiology, %V 1 %N 1A %@ 2516-8290 %C 213 %R https://doi.org/10.1099/acmi.ac2019.po0082 %I Microbiology Society, %X Antimicrobial resistance (AMR) is a growing threat to healthcare. Many AMR-encoding genes are carried on accessory genetic elements like plasmids, which spread between phylogenetically distant members of bacterial communities. CRISPR-Cas based technologies may help combat the spread of AMR genes by removal of such plasmids. To optimise this technology, implementation of a delivery method which can reach a diverse range of bacteria is needed. Therefore, we engineered broad host-range conjugative plasmid pKJK5 to express Cas9. pKJK5::Cas[GmR] encodes a guide RNA which targets Gentamicin resistance gene aacC1, while pKJK5::Cas[nt] encodes a non-targeting guide RNA. After confirming its Ca9 activity by electroporation of targeted and untargeted plasmids, we set up a proof-of-concept experiment to conjugatively deliver pKJK5::Cas from a donor Escherichia coli strain DH5α to unrelated recipient E. coli K12, and to remove Gentamicin-resistance encoding target plasmid pHERD30T. For these means, we mixed donors and recipients in liquid media and incubated them overnight; proportions of the total population were enumerated by differential plating. We found that Gentamicin-resistant recipients were reduced by 33 % when treated with pKJK5::Cas[GmR] compared to treatment with pKJK5::Cas[nt], indicating that pKJK5::Cas[GmR] has the ability to remove AMR plasmids from recipient cells. This proof-of-concept experiment shows how an engineered broad host-range conjugative plasmid is an effective means of removing AMR-encoding plasmids and may be a viable approach to remove resistance genes from complex bacterial communities. To make this method more effective, community experiments as well as optimisation of Cas9 activity are needed. %U https://www.microbiologyresearch.org/content/journal/acmi/10.1099/acmi.ac2019.po0082