CRISPR-based antimicrobials to obstruct antibiotic-resistant and pathogenic bacteria.

Abstract

The rapid emergence of antibiotic-resistant bacteria and the comparatively limited development of new antibacterial molecules pose a major threat to modern medicine by jeopardizing our ability to treat and prevent infections [1]. The overuse of antibiotics in both medical and agricultural settings has facilitated the emergence of multidrug-resistant (MDR) bacteria [2]. Furthermore, many antibiotics lack specificity, indiscriminately killing pathogenic and nonpathogenic bacteria and contributing to antibiotic-associated infections [3,4]. This highlights the critical need for novel therapeutics that circumvent existing modes of drug resistance while adding specificity [5]. Toward this goal, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) systems are being explored as possible antimicrobials to target bacteria and/or antibiotic resistance genes in a sequence-specific manner. These promising targeted therapies are the focus of synthetic biology companies such as Locus Biosciences, Intellia Therapeutics, and Eligo Bioscience, to name a few. Although these technologies are mostly in the preclinical phase, the main goal is to provide precision antimicrobials for infectious diseases. Here, we discuss the present state of CRISPR antimicrobial research, with a specific focus on the biological differences between targeting plasmids versus chromosomes, the efficacy of CRISPR antimicrobials in infection and colonization models, and the various delivery mechanisms for these potential therapeutic tools. Sequence-specific targeting by CRISPR/Cas CRISPR/Cas systems are adaptive defense mechanisms against mobile genetic elements (MGEs). A key hallmark of these systems is the CRISPR array, a genomic locus comprised of unique spacers alternated by identical repeats [6]. Effector proteins function as interference molecules to silence foreign genetic elements [6]. During adaptation, some CRISPR types integrate a short fragment of foreign DNA into the CRISPR array, thereby providing a genetic memory of MGE invasion, referred to as a spacer (Fig 1). Transcription of the CRISPR array yields precursor CRISPR RNAs (pre-crRNAs) that are enzymatically processed into mature crRNAs (Fig 1). Upon invasion of bacteria by MGEs with complementary sequence, crRNAs guide effector proteins to these targets for enzymatic cleavage, ultimately causing sequence-specific elimination of the invading molecule [6] (Fig 1). Open in a separate window Fig 1 Mechanisms of action of CRISPR/Cas immunity. Left: Class I CRISPR/Cas systems (modeled by Type I system) utilize a multi-subunit complex, termed “Cascade,” as the effector machinery. Right: Class II CRISPR/Cas systems (modeled by Type II system) use a single effector protein (such as Cas9) for interference. Both classes also consist of spacer (diamonds) and repeat (squares) arrays. Top: During adaptation, the Cas1–Cas2 complex takes a sequence of the invading DNA and integrates it into the CRISPR array as a novel spacer. Center: In the next stage, termed “expression,” the CRISPR array is transcribed into pre-crRNAs that are further processed into mature interfering crRNAs. Bottom: During the interference stage, the mature crRNAs guide the Cas proteins to their DNA target. Upon binding of the crRNA to their cognate DNA target, the Cas protein generates a double-stranded DNA break in the target. Created with BioRender.com. Cascade, CRISPR-associated complex for antiviral defense; CRISPR/Cas, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein.