<b>Engineering probiotic</b><b> </b><b>Escherichia coli</b><b><i> </i></b><b>Nissle 1917 to block transfer of multiple antibiotic resistance genes by exploiting a type I</b><b> </b><b>CRISPR-Cas</b><b> system</b>

Many multidrug-resistant (MDR) bacteria have evolved through accumulation of antibiotic resistance genes (ARGs). Although the potential risk of probiotics as reservoirs of ARGs has been recognized, strategies for blocking transfer of ARGs while using probiotics have rarely been explored. The probiotic Escherichia coli Nissle 1917 (EcN) has long been used for treating intestinal diseases. Here, we showed frequent transfer of ARGs into EcN both in vitro and in vivo, raising its potential risk of accumulating antibiotic resistance.<i> </i>Given that no CRISPR-Cas system was found in natural EcN, we integrated the type I-E CRISPR-Cas3 system derived from E. coli BW25113 into EcN, and showed that the engineered EcN was able to efficiently cleave multiple ARGs (i.e., mcr-1, blaNDM-1 and tet(X)) encoding enzymes for degrading last-resort antibiotics. By co-incubation of EcN expressing Cas3-Cascade and that expressing Cas9, we showed that the growth of the former strain outcompeted the latter strain, demonstrating better clinical application prospect of EcN expressing the type I-E CRISPR-Cas3 system. In the intestine of a model animal (i.e. zebrafish), the engineered EcN exhibited immunity against transfer of CRISPR-targeted ARGs. Our work equipped EcN with immunity against transfer of multiple ARGs by exploiting the exogenous type I-E CRISPR-Cas3 system, thereby reducing the risk of spread of ARGs while using it as probiotic chassis for generating living therapeutics.