Advancing the design and delivery of CRISPR antimicrobials
Graphical abstract
Introduction
CRISPR-Cas systems are adaptive immune systems in bacteria and archaea whose CRISPR nucleases and guide RNAs have formed the foundation of powerful tools in biotechnology and medicine. The guide RNAs direct the nuclease to bind and cleave complementary DNA or RNA sequences often flanked by a protospacer-adjacent motif (PAM). The ease of designing guide RNAs, the portability of the nuclease-guide RNA pair, and the broad applicability of programmable DNA binding and cleavage has given rise to a diverse set of applications ranging from genome editing and gene regulation to in vitro diagnostics, real-time DNA and RNA imaging, and gene drives 1, 2.
The standard mechanism of CRISPR-based genome editing is the directed repair of cleaved DNA. In eukaryotic cells, the cleaved DNA is efficiently repaired through ubiquitous mechanisms such as homology-directed repair or non-homologous end joining. However, in bacteria, multiple studies have indicated that DNA cleavage by CRISPR nucleases often cannot be repaired and is therefore lethal. One line of evidence is the dearth of naturally occurring genome-targeting spacers in active CRISPR-Cas systems, even though incorporation of such sequences regularly occurs during naive spacer acquisition 3, 4. Separately, the introduction of genome-targeting guide RNAs in bacteria with an active CRISPR-Cas system arrested the cell cycle, leading to eventual death or loss of large genomic segments containing the target sequence 5, 6. These insights helped inspire the use of CRISPR nucleases as programmable antibacterial agents that could programmably and irreversibly destroy targeted DNA only in selected bacteria. This mechanism has been developed primarily to address the rising challenge of antibiotic resistance and the diminishing supply of new small-molecule drugs [7]. Here, we review how CRISPR nucleases have been harnessed as programmable antimicrobial agents to combat human disease and the rise of antibiotic resistance, and we discuss opportunities for the further development of this promising antimicrobial strategy.
Section snippets
CRISPR nuclease selection
CRISPR-Cas systems commonly act as immune systems by cleaving foreign DNA or RNA, yet a diversity of system types have been reported (Figure 1A) 8, 9, 10. These system types are differentiated based on the associated proteins responsible for acquisition and targeting as well as their modes of action. For instance, Type II systems generate blunt-ended cuts in target DNA, Type III systems synergistically cleave target RNA and DNA, and Type VI systems cleave target RNA followed by the
CRISPR antimicrobial delivery
Arguably the most pervasive challenge to the development of CRISPR antimicrobials is efficient delivery to diverse bacteria. To-date, CRISPR antimicrobials have been principally encoded as DNA and delivered using bacteriophage particles ∗∗20, 21, ∗22, ∗∗27 (Figure 2A). Bacteriophage (or phage for short) are adept at injecting their genetic material across the bacterial cell wall and into the cytoplasm of its host. CRISPR antimicrobials have been encoded in two forms to ensure packaging by the
Future challenges and opportunities
Even if DNA encoding a CRISPR antimicrobial is efficiently delivered to the target cell, the DNA must avoid the host's immune systems to allow sufficient antimicrobial expression. Different types of immune systems are known, such as restriction-modification, CRISPR-Cas, and abortive infection among others, and the specific barriers can vary widely even between related bacteria. Some of these barriers can be overcome through common techniques that have been developed through efforts to improve
Conclusions and future perspectives
CRISPR antimicrobials represent one of many uses of CRISPR-derived technologies. The CRISPR antimicrobial field is still in its infancy and faces numerous challenges for the design and delivery of these antimicrobials. We also envision future hurdles as CRISPR antimicrobials move from the lab to animal models and finally to human trials. Specifically, further investigation and engineering is needed to ensure that the phage delivery vehicles reach the site of infection and efficiently deliver
Acknowledgements
This work was supported by the Bill and Melinda Gates Foundation [grant number OPP1140021], the North Carolina Biotechnology Center [Technology Enhancement Grant], and the Bay Area Lyme Foundation [Emerging Leader Award]. C.L.B. is a co-founder of Locus Biosciences and is an inventor on patent applications related to CRISPR technologies.
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