Review Driving to Safety: CRISPR-Based Genetic Approaches to Reducing Antibiotic Resistance Ethan Bier 1,2, * and Victor Nizet 1,3,4 Bacterial resistance to antibiotics has reached critical levels, skyrocketing in hospitals and the environment and posing a major threat to global public health. The complex and challenging problem of reducing antibiotic resistance (AR) re- quires a network of both societal and science-based solutions to preserve the most lifesaving pharmaceutical intervention known to medicine. In addition to developing new classes of antibiotics, it is essential to safeguard the clinical efficacy of existing drugs. In this review, we examine the potential application of novel CRISPR-based genetic approaches to reducing AR in both environmental and clinical settings and prolonging the utility of vital antibiotics. The Antibiotic Resistance Crisis Since their introduction, antibiotics have reduced human mortality rates from infectious diseases by 80% [1]. Unfortunately, antibiotic resistance (AR) among leading bacterial pathogens is currently estimated to cost >700 000 lives annually [2], nearly equal to the mortality attributed to all the world’s most deadly mosquito-borne diseases combined i [3]. Widespread overprescrip- tion of antibiotics and their misuse in animal husbandry have increased the prevalence of AR in medical facilities [4] and in the environment [5–7]. Evidence indicates that environmental sources of AR are transmitted via bacterial intermediates to human populations and contribute signifi- cantly to the current health crisis of antibiotic treatment failures in resistant infections ii [6,8,9]. As troubling as the current situation is, health experts predict that AR threats could markedly worsen in the coming decades iii [10], leading to some 10 million AR disease deaths per year by 2050 if left unchecked [2]. This ballooning crisis can only be addressed by synergistic efforts to develop strict new antibiotic stewardship guidelines by the medical establishment [11]; legislation to prohibit inappropriate agricultural practices, such as adding antibiotics in animal feed to enhance livestock growth; and robust partnerships spanning academia, industry, philanthropies, and government agencies to develop new natural or synthetic antibiotics [12], innovative immu- notherapies [13], or novel antibacterial [14] and anti-AR compounds [15,16] to extend the longevity of existing antibiotics. CRISPR-Based Strategies to Combat AR The discovery of a bacterial immunity system referred to as CRISPR (clustered regularly interspaced short palindromic repeats; see Glossary) has given rise to a revolution in pre- cision genetic engineering in both prokaryotic and eukaryotic organisms [17,18]. Among this ever-expanding array of immune recognition and protective mechanisms, type II CRISPR systems, the best studied and most widely applied to practical ends, include both protein (e.g., Cas9) and RNA [e.g., endogenous cRNAs and trans-activating CRISPR RNAs (tracrRNAs), and synthetic guide RNAs that fuse the cRNAs and tracrRNAs into a single tran- script, referred to hereafter as gRNAs], which form ribonucleotide–protein complexes that cut DNA bases at sites complementary to a 20–base pair target recognition sequence in the gRNA (Figure 1A). Highlights Synthetic CRISPR systems have been developed to combat antibiotic resistance (AR). Phage and conjugative horizontal gene transfer vehicles can dissemi- nate CRISPR anti-AR platforms throughout bacterial populations. Anti-AR CRISPR systems may reduce AR prevalence in experimental infection models. Self-amplifying proactive genetic sys- tems increase anti-AR efficiency approxi- mately 100-fold. Guide RNA–directed transposons should allow insertion of anti-AR CRISPR platforms into multiple defined genomic or episomal target sites. 1 Tata Institute for Genetics and Society, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0349, USA 2 Section of Cell and Developmental Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0349, USA 3 Collaborative to Halt Antibiotic- Resistant Microbes, Department of Pediatrics, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0687, USA 4 Skaggs School of Pharmacy & Pharmaceutical Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0687, USA *Correspondence: ebier@ucsd.edu (E. Bier). Trends in Genetics, Month 2021, Vol. xx, No. xx https://doi.org/10.1016/j.tig.2021.02.007 1 © 2021 Elsevier Ltd. All rights reserved. Trends in Genetics TIGS 1791 No. of Pages 13