Abstract
Abstract
In the arms race of bacteria and phages, bacteria employ immune responses to defend themselves against phage invasion, while phages, in turn, develop counteractive strategies to bypass bacterial defenses. In a Nature publication, Camara-Wilpert etal. shed light on a previously unknown mechanism employed by viruses to thwart bacterial antiviral defenses, which opens a new avenue for inhibiting clustered regularly interspaced palindromic repeats (CRISPR)-Cas genome editing in eukaryotes as well as offering spatial, temporal, and conditional control over the process.
The primary objective of the clustered regularly interspaced palindromic repeats (CRISPR)-Cas system is to confer adaptive immunity to prokaryotes against viruses and several mobile genetic elements (MGEs). These nuclease complexes are broadly classified into six types (I–VI), two main classes (class 1: multiprotein; I, III, and IV and class 2: single protein; II, V, and VI), and more than 30 subtypes based on their genetic compositions and interference mechanisms.1 The CRISPR-Cas system demonstrates high specificity and robust programmability, rendering it indispensable for various biotechnological and therapeutical applications, such as genome editing, gene regulation, and gene knockouts. Despite its potential applications, several challenges persist, including Cas nuclease-based off-target effects, cellular toxicity, unintended on-target effects, and immunogenicity.
The immune response of bacteria and archaea has executed in three distinct stages, i.e., adaptation, processing, and interference (Fig.1a). This orchestrated process serves as a potent defense mechanism against MGEs, including phages and plasmids. On the contrary, MGEs have overcome the bacterial immunity by evolving anti-CRISPR (Acr) proteins (also known as “off switches and natural brakes”). To date, approximately 100 Acr protein families have been discovered, exhibiting the capacity to inhibit types I, II, III, V, and VI CRISPR-Cas systems at multiple stages.2 Notably, these Acr proteins demonstrate substantial difference in mechanisms and structures, displaying no sequence similarities among them. The unique capability of Acr proteins is to disrupt CRISPR functions in the heterologous hosts and offers genetically encodable and post-translational regulation for CRISPR-Cas-based tools. For instance, inhibitors such as AcrIIC1 and AcrIIC3 targeting NmeCas9, as well as AcrIIA2 and AcrIIA4 targeting SpyCas93 have successfully demonstrated their ability to regulate CRISPR-Cas function in human cells. Most identified Acr proteins inhibit or bind to Cas proteins, disrupting their interactions, biochemical activities, and playing a crucial role in structural transitions (Fig.1b).
Recently, Camara-Wilpert etal. reported a new CRISPR-Cas inhibition strategy based on the non-coding RNA anti-CRISPRs (Racrs). They identified Racrs encoded by viral genomes as solitary repeat units (SRUs), which mimics bacterial crRNAs and effectively divert the antiviral defense into a dead-end path. These SRUs consist of one spacer and one repeat, and unlike most viral CRISPR arrays are not associated by genes encoding Cas proteins. The authors unveiled these SRUs and hypothesized that they have Acr functions. To test this hypothesis, they found SRUs, identical to crRNAs encoded by their selected bacterial strain and integrated SRU into that bacterial strain. Surprisingly, they observed that the SRUs expressed crRNA-like small RNAs, while previously bacteria targets and suppress the phage, by type 1-F CRISPR-Cas system. The authors demonstrated that RacrIF1 inhibits type I-F CRISPR-Cas system by interacting with Cas6f and Cas7f, leading to the formation of a Cas subcomplex. Mutation in racrIF1, preventing the binding of Cas6f with RacrIF1 RNA, impacted Acr activity. Furthermore, the authors proposed that RacrIF1 functions as a crRNA mimic, forming an aberrant complex upon binding with Cas6f. This interaction sequesters Cas proteins, preventing their participation in antiviral defense (Fig.1c). Additionally, they suggested that phages and other MGEs expressing SRUs can encode Racrs, acting as inhibitors of CRISPR-mediated defense. They identified several SRUs in phages and other MGEs across diverse species, displaying similarities to crRNAs. Additionally, the team successfully found Racr candidates for nearly all CRISPR-Cas types, and when these candidates were expressed in bacteria, it was demonstrated that nine of them possess Acr function. Furthermore, there is a tendency for racr genes to cluster together with acr genes inside viral genomes, indicating that they have similar functions. Moreover, the authors utilized a high-throughput computational approach to identify Racrs across the diverse taxa of MGEs that infect prokaryotes. This showed the rapid evolutionary mechanism of MGEs and suggests that similarly novel and vital CRISPR inhibitory mechanisms are waiting to be discovered.4
The Acr proteins have numerous limitations such as cell permeability issues, instability, and immunogenicity.5 Additional research is required to validate whether Acr proteins serve as safe and efficient “off switches” invivo, whereas small non-coding Racrs have been identified as competitive Acr molecules that can impede RNP formation and affect DNA/RNA binding, exhibiting promising designs. Furthermore, the identification of new Acr proteins can be complex and long drawn out, but the strategy employed by Camara-Wilpert etal.4 in identifying Racrs similarities to known CRISPR repeats has great potential for enabling rational design strategies. Moreover, the authors also identified that racr genes tend to cluster with acr genes within viral genomes, demonstrating their functional equivalence. The advent of broad-spectrum Racrs introduces an additional layer to modulate genome editing in eukaryotes. The authors' findings strongly indicate that Racrs are a common and unexplored mode of CRISPR counterdefense. The novel mimicry strategy employed by phages enhances our understanding and paves the way for the development of new CRISPR-Cas systems.
Biotechnological Applications of Acrs
With the increasing demand for CRISPR-Cas applications in medicines, synthetic biology, and agriculture, there is an urgency to discover new CRISPR-Cas inhibitors for controlling devastating genome engineering effects. CRISPR-Cas-based genome editing is now successfully tested to precisely induce large deletions (up to 304 kb)6,7 to large insertions (up to 36 kb),8,9 offering unprecedented opportunities to improve health, agricultural, and environmental sectors. However, safety concerns persist, both due to genotoxic effects of on-target double-stranded DNA breaks (e.g., induction of p5310) and off-target editing activity. To reduce these unintended edits, one of the most prominent approaches is the utilization of Acr proteins, which may have the ability to restrict CRISPR genome editors in both bacterial and eukaryotic cells.11
Following are the potential examples of Acrs utilized to improve CRISPR-Cas effectiveness and safety concerns: (1) Acrs may suppress the toxicity caused by CRISPR-Cas genome editors during the modification of bacterial genomes.12 Additionally, its delivery further reduces the cytotoxicity of CRISPR-based genome editing in human cells, exhibiting a flexible and tunable method for limiting off-target effects; (2) Another important application of CRISPR inhibitor proteins lies in the field of CRISPR-Cas-based gene drive, which has the potential to disseminate engineered traits within a population through a super-Mendelian mechanism. Gene drive holds promise for benefiting human health in several ways, including curtailing devastating insect-borne diseases and controlling agriculturally important insect-pest. However, the application of gene drive in the wild carries the risk of unforeseen consequences or potential misuse.13 Recently, Basgall etal. showed that Acr proteins (AcrIIA2 and AcrIIA4) could halt gene drives at multiple stages in yeast with an efficiency of >99.9%.14 However, the efficacy of Acr proteins for halting gene drives or off-target editing in animals also remains to be determined specifically, whether mating a gene drive animal with an Acr animal returns inheritance to normal Mendelian frequencies or results in unforeseen outcomes; (3) other potential applications include reversing the effects of dCas9 binding to a genomic locus15 or limiting the duration of Cas9 activation in the nucleus to reduce off-target gene editing.16 dCas9 has been utilized to enhance or suppress the transcription of several genes, to modify epigenetic marks, and to localize fluorescent labels used for imaging of different loci,15 whereas to limit the off-target effects, AcrIIA4 was employed to halt DNA binding by a dCas9 fusion to Tet1, which is a DNA methyl modification enzyme, in induced pluripotent cells.17 Thus, Acrs of Cas9 have the potential to improve the limitations of unintended off-targets effects as well as safety concerns of Cas9-mediated technologies; (4) The Acrs can also be used to precisely adjust gene expression levels for a range of biotechnological purposes, such as synthetic biology and metabolic engineering. For instance, Nakamura etal. presented a proof-of-concept pulse generator circuit using Acr-dCas9 interaction, indicating potential for Acrs in quantitative synthetic biology, potentially integrating Acrs and dCas9 effectors into gene regulation programs18; (5) Another application of Acrs is to detect the CRISPR-Cas effector complex. To assess the effectiveness of CRISPR-Cas gene editing in specific cells, it is necessary to identify the presence of the CRISPR-Cas effector complex in biological samples. Acr-based biosensing technologies provide an alternative to antibodies for detecting, identifying, and quantifying effector complexes owing to the strong binding affinity of Acrs for effector complexes.19 In a nutshell, Acr proteins possess numerous potential applications, such as identifying enduring epigenetic modifications, precisely detecting CRISPR-Cas complexes, serving as a valuable tool in phage therapy to achieve CRISPR resistance, limiting editing activity to different tissues or developmental stages, improving gene-editing strategies, and reducing the harmful effects of genome editing.
Overall, the intricate dynamics of the evolution of bacterial immune responses against phage predation and the neutralization of bacterial immune responses by Acr proteins will not reach a stalemate. Instead, it serves as a signal to remain vigilant for the emergence of anti-Acr mechanisms, marking the next phase in this dynamic molecular arms race. Understanding these intricate interactions will be crucial for advancing our control over CRISPR-based technologies and their applications. Exploring phage-bacteria arms race holds the key to identifying novel off-switches that can be leveraged in genome editing as well as a range of other biotechnological and therapeutical applications.