Bacteria and their viruses (phage) have been engaged in a never-ending weapon race for over 3 billion years. Bacteria are a human microbiome component, making them more intricately linked to people than other organisms. Numerous bacteria are able to coexist with people without causing harm due to the establishment of mutualistic relationships; nevertheless, some of these bacteria are pathogenic (Berg et al., 2020). Bacteriophages, on the other hand, are ubiquitous in the microbial world. There are currently more than a thousand phages that attack a bacterium each and every day (Knott and Doudna, 2018). Restriction modification, superinfection exclusion, an abortive infection mechanism, and other activities make up the anti-phage defensive system, which helps bacteria fend off viruses and other invaders. That is all part of the innate immunity of the bacterial defense system. Bacteria also contain the CRISPR-Cas system, an adaptive immunological defense mechanism (Abedon, 2012). CRISPR-Cas is a self- and non-discriminatory system that is found in Archaea and Bacteria. It is responsible for the development of an adaptive defense mechanism in these organisms by combining highly specialized nucleases with genetic memory (Eitzinger et al., 2020). CRISPR is named after the sequence’s actual nature, which is “clustered regularly interspaced short palindromic repeats”. Prokaryotes use CRISPR and its associated protein (Cas9) as part of their adaptive immune response to viruses and bacteriophages (Hille and Charpentier, 2016).

The CRISPR defensive system consists of three fundamental stages: adaption (spacer acquisition), crRNA synthesis (expression), and target interference. These stages work together to protect bacteria from multiple viral attacks (Hille and Charpentier, 2016). CRISPR loci are clusters of regularly interspaced short repeats that can be found in the chromosomal or plasmid DNA of certain prokaryotes. The Cas gene, which encodes the nuclease protein (Cas protein) responsible for destroying or cleaving viral nucleic acid, is typically located next to the CRISPR gene (Asmamaw and Zawdie, 2021). The Cas proteins detect the foreign invading DNA, cut it up, insert it into the CRISPR spacer area, and store the fragments in the genome (adaptation). The transcription of the CRISPR region results in the production of pre-crRNA, which is then processed into smaller RNA units called CRISPR RNA (crRNA) (expression). crRNA’s spacer sequence homology aids in the capture of foreign DNA, which is then cleaved by a complex containing the nuclease-active Cas protein (Interference) (Sontheimer and Davidson, 2017; Ishino et al., 2018). Because of their role, crRNAs are also known as guide RNAs (gRNAs) (Brouns et al., 2008). Short viral DNA fragments (spacers) are inserted into the CRISPR array of a bacterial cell during the adaptation process, immunizing the cell against the virus. Thus, spacers act as a genetic memory of earlier viral infections. This is how the CRISPR region remembers previously invaded bacteriophages or viruses and passes down their molecular memory from generation to generation (Ran et al., 2013).

The Red Queen hypothesis postulates that, in order for species to avoid extinction, they will need to continually evolve new mechanisms of resistance to parasites (Brouns et al., 2008). In response, the parasites develop their own countermeasures, which allow them to evade the resistance mechanisms. This battle for survival can be traced back to the co-evolutionary dynamics of bacterial populations and bacteriophages (Pawluk et al., 2018). Through counter defense evolution in the microbial world, phages have evolved the inhibitors of CRISPR-Cas systems. The first known CRISPR-Cas inhibitors were found in a family of phages from the genus Pseudomonas spp. Although they had protospacer sequences that should have been targeted by this system, these phages were, nonetheless, able to infect and multiply in a Pseudomonas aeruginosa strain with an active type I F CRISPR-Cas system (Bondy-Denomy et al., 2013). Phages encode anti-CRISPR (Acr) proteins to evade the CRISPR-Cas immune system of their hosts (Maxwell, 2017). Acr proteins invade viral DNA into bacteria, shielding the phage’s genetic material from the CRISPR system (Semenova et al., 2011). Acr proteins have been studied structurally and biochemically, and their inhibitory effects have been found to encompass a broad array, from blocking crRNA loading to preventing target DNA recognition and DNA cleavage (Davidson et al., 2020; Pinilla-Redondo et al., 2020). To date, there have been 98 unique families of Acr proteins described against CRISPR-Cas systems. The search for new Acr has been aided by the development of bioinformatics tools such as AcRanker, AcrFinder, and PaCRISPR and online databases like Anti-CRISPRdb, AcrDB, AcrHub, and AcrCatalog (Bowen et al., 2022).

CRISPR-Cas is being used for genome engineering in plants and programmable gene regulation, highlighting the necessity for control mechanisms for its various activities (Calvache et al., 2022). Acr proteins, in this light, are an underutilized regulatory mechanism in plant biotechnology. In both herbaceous and woody plant species, Acr proteins are effective at inhibiting CRISPR-Cas9–based genome-editing tools (Liu et al., 2023). In this review, the numerous types of Acr proteins, their respective mechanisms of action, and the broad range of applications were discussed. In contrast to earlier studies, this review includes the most recent data on Acr proteins, their uses, and a detailed explanation of their mechanisms of action.

CRISPR-Cas is a revolutionary targeted genome-editing approach that can modify a genome from any region of any species with great precision and accuracy without causing harm to other genes (Koonin and Makarova, 2019). CRISPR loci serve as a genetic library, consisting of two parts: the first is the CRISPR array, which represents the immunological memory primarily derived from foreign genetic elements encoded within individual spacers separated by a gap (Koonin and Makarova, 2019) and the second is crRNA, coupled with one or more Cas proteins in the resulting CRISPR-cas complex, which identifies the invading foreign genetic material. The protospacer, which is frequently flanked by a protospacer neighboring motif, is the location of invading DNA targeted by the spacer [proto-spacer adjacent motif (PAM), usually 2–4 nucleotides]. PAM sequences are important in most circumstances because they help the cell to distinguish between its own DNA and invading foreign DNA. For instance, when foreign DNA gets in the proximity of crRNA, it forms the complex with Cas protein followed by cleavage and destruction of foreign DNA (Zhang, 2019). The CRISPR-Cas system is highly diverse, and, based on the complement of unique Cas genes, length of spacers, and difference in palindromic repeats, the CRISPR-Cas system is classified into two classes: six subclasses (types I–VI) and numerous sub-subclasses (subtypes). The CRISPR-Cas system is divided into two classes based on the functions and structure of Cas-protein (CRISPR associate protein) (Makarova et al., 2015; Mohanraju et al., 2016; Zhang, 2019). The class 1 systems include a multi-subunit Cas protein complex (types I, III, and IV) to recognize and cleave the nucleic acid. The class 2 system (types II, V, and VI) includes large and multifunctional Cas proteins, for example, Cas9 that participates in DNA targeting and cleavage (Zhang, 2019). In recent years, the development of CRISPR-based genome-editing technologies has revolutionized the fields of molecular biology and genetics (Maxwell, 2017). CRISPR-Cas technology, as a genome-editing tool, is tremendously useful in preventing genetic disease and fighting genetic abnormalities (Maxwell, 2017). Furthermore, by generating genomes that are more environmentally friendly, the CRISPR-Cas technique is being used to improve the quality of valuable commercial plants and to find solutions for a number of hereditary disorders.

Bacteria use the most dependable immune system, CRISPR-Cas9, to defend themselves against phage attacks. Prior to the discovery of the Acr protein, the only option for phages to avoid CRISPR-Cas–mediated destruction was by point mutations (Loureiro and Da Silva, 2019). Changes in their sequences make bacteria unable to recognize the phage genome, allowing phages to live for longer periods of time. However, they are only protected until the bacteria reproduce their new sequence with mRNA and insert it into the CRISPR area. Acr proteins, which block the CRISPR-Cas system, have evolved in phages to circumvent CRISPR-Cas–mediated immunity (Maxwell, 2017). Infection with a CRISPR-targeted phage in the presence of P. aeruginosa prophage was monitored using a functional test (Jooyoung et al., 2018), Bioinformatic linkages with known aca genes and Acr proteins “guilt by association”, self-targeting within the same genome, were used in bioinformatic investigations to identify latent Acr candidates in prophages. The lytic phage is being screened for those who do not receive an immune response from the CRISPR system (Maxwell, 2017).

Acrs are bacteriophage genes that were formerly recognized as auxiliary genes (Juhala et al., 2000). Although these accessory genes are not required for the phage’s life cycle, their presence among phages suggests that they provide an evolutionary benefit in some circumstances (Harald et al., 2004). Recently, it was discovered that the CRISPR-Cas defensive system is only present in 50% of bacteria, in which case, Acr proteins are inappropriate and apparent Acr proteins are absent. Acr proteins are also known as inhibitor proteins because they stop the CRISPR-Cas system from destroying the phage genome. In 2013, the first Acr protein was discovered in a collection of closely related phages that infect Pseudomonas aeruginosa (Bondy-Denomy and Davidson, 2014). They permitted a phage that should have been targeted and destroyed by the type I-F CRISPR-Cas system to infect and kill the bacterium after integrating these phages into the bacterial genome (a form known as prophage). In P. aeruginosa, four additional Acr proteins (Acr IE1–4) were discovered to block the I-E CRISPR-Cas system (Jooyoung et al., 2018). As a result of applying the guilt-by-association strategy, the first inhibitors of the type II CRISPR-Cas system were found. Three Acr protein families—AcrIIC1, AcrIIC2, and AcrIIC3—were found to block the activity of type II-C CRISPR-Cas9 in Neisseria meningitides and were the first Acr proteins used for Cas9-mediated genome editing in human cells (Pawluk et al., 2016a). Pawluk and their team used a lytic phage technique to identify AcrIIA5 and AcrIIA6 proteins in two virulent phages, and AcrIIA5 has proven to be the most broad-spectrum inhibitor of the type II CRISPR-Cas proteins. Although a large number of Acr proteins have been found ( Table 1 ), only a handful have been thoroughly described until now. AcrIIA1, AcrIIA4, AcrFI, AcrFII, AcrF3, and AcrF10 are the most studied Acrs.

The CRISPR-Cas9 system has swiftly evolved into a useful tool for improving the genomes of plant species. Plant quality has been enhanced through the manipulation of genes to alter metabolic pathways, to increase stress resistance (Os8N3, OsProDH, OsGS3, and OsNAC45 genes in Rice) (Usman et al., 2021), to increase disease resistance (MdDIPM4 gene in apple; EgIFR gene in Oil palm) (Gan and Ling, 2022), to improve flowering time and plant height (ZmPHYC1 ZmPHYC2 gene in maize) among other things (Li et al., 2020; Montecillo et al., 2020). Increased oleic acid content, higher amylose content, and a thermo-sensitive genic male sterile line are all results of the CRISPR-Cas9 system’s work with Oryza sativa (Sun et al., 2017; Abe et al., 2018; Zhou et al., 2019). The CRISPR-Cas9 system, in a similar manner, confers biotic and abiotic stress resistance or tolerance in plants, for example, including resistance in Cucumis sativus to potyviruses such as Zucchini yellow mosaic virus and Papaya ring spot mosaic virus-W (Chandrasekaran et al., 2016) and immunity in Cucumis sativus to Cucumber vein yellowing virus (Ipomovirus) infection, drought resistance in Zea mays (Shi et al., 2017), and salinity tolerance in rice (Zhang, 2019).

In the long term, it was unknown how phages were able to successfully infect bacteria until Acr proteins were discovered in Pseudomonas aeruginosa. Acr proteins can impede the formation of new CRISPR spacers, block cas protein production, obstruct crRNA transcription, prevent the active CRISPR-Cas complex from forming, inhibit binding to foreign DNA elements, and block cleavage activity (Maxwell, 2017). Disruption of DNA binding, inhibition of target sequence, enzymatic degradation of secondary messenger signaling molecule, and cleavage are the four common mechanisms used by Acr proteins (Jia and Patel, 2021). Over the last few years, genetic, biochemical, and structural studies have been used to determine the mechanism of activity of Acr protein families (Pawluk et al., 2016b). Acr proteins do not disrupt the expression of the Cas gene or the process of crRNA but disrupt the DNA binding activity through direct interaction with the CRISPR-Cas complex. In addition, Acr prevents DNA from attaching to the viral genome, allowing it to evade detection (Maxwell, 2017). P. aeruginosa type 1-F Acr proteins AcrF1, AcrF2, and AcrF3 have been found to interact directly with the type 1-F CRISPR cascade complex, inhibiting its affinity for DNA binding ( Figure 1 ) (Bondy-Denomy et al., 2015). AcrF1 attaches to Cas7F as two to three copies of each monomeric unit in the hexameric unit. Cas7f is more commonly referred to as the type 1-F cascade complex’s backbone (Bondy-Denomy et al., 2015). A key contact is created between a cluster of three essential residues on the surface of AcrF1 and exposed lysine residues in the Cas7F protein backbone, which inhibits DNA targeting access (Bondy-Denomy et al., 2015; Maxwell, 2017). AcrF2 is an acidic protein that acts as a DNA mimic and sterically prevents DNA binding via an interaction with the Cas8f-Cas5f heterodimer (Bondy-Denomy et al., 2015). For example, the Acr protein AcrIIA4 binds with the PAM. It is an interacting area of the type 2-A Cas9/Single Guide RNA (sgRNA) complex that keeps the target DNA sequence from getting in the way (Chowdhury et al., 2017). X-ray crystallography and cryo-electron microscopy were utilized to examine these interactions which revealed that AcrF3 binds to the interaction interface, blocking the Cas3 binding site for DNA and locking Cas3 in ADP-bound form. Similarly, type 2-C AcrIIC1 prevents the Cas9 HNH endonuclease from cleaving both target and non-target DNA strains. Potentially, only a few Acr protein structures and mechanisms have been determined to date, and many possible effective strategies may occur with viral Acr gene families (Wang and Wang, 2017).

The versatile and effective use of the CRISPR-Cas system can be utilized for various applications, such as gene editing and chromatin imaging in eukaryotic cells (Jinek et al., 2012; Mali et al., 2013). Spatial and temporal investigations of chromatin dynamics are now possible because of the combination of live imaging of chromatin with programmable DNA binding proteins developed through genome-editing techniques (Fujimoto and Matsunaga, 2016). Acr proteins can be employed in novel ways to regulate CRISPR-Cas function because of their distinctive methods of action. Acrs can be used to enhance the activity of the CRISPR-Cas system in both prokaryotes and eukaryotes. The capability of numerous Acr proteins to directly interfere with CRISPR-Cas functions in heterologous hosts allows genetically encodable, post-translational regulation for technologies generated from CRISPR-Cas. Evaluation of the efficacy of CRISPR-Cas–based gene editing in target cells requires the detection of the CRISPR-Cas effector complex within biological materials. Acr-based biosensing technologies offer an alternative to antibodies for effector complex detection, identification, and quantification due to the high binding affinity of Acrs for effector complexes (Kaminski et al., 2021). The use of phage therapy and the use of bacterial viruses (phages) to treat bacterial infections are increasingly being studied as a potential replacement for antibiotics (Brives and Pourraz, 2020). The use of Acrs such as Acr IIA4, AcrVIA2, AcrVIA5, AcrIIA5, and AcrIIA2 is a proven method for minimizing the target’s effects of CRISPR-Cas tools in various hosts (Liang et al., 2020; Jia and Patel, 2021). Several research has provided evidence that Acrs such as AcrIIA4, AcrIIC1, and AcrIIC3 can be used to achieve cell-specific control of the gene-editing process carried out by CRISPR-Cas9 (Lee et al., 2019; Liang et al., 2020; Jia and Patel, 2021). Timed administration of AcrIIA2 and AcrIIA4 was shown to regulate CRISPR-Cas9 activity, decrease the cytotoxicity of human hematopoietic stem cells, and boost engraftment rates without impairing on-target genome editing (Jia and Patel, 2021). In Saccharomyces cerevisiae, it was discovered that the proteins AcrIIA2 and AcrIIA4 were able to deactivate Cas9, making them powerful gene drive inhibitors. Acr proteins have many potential applications, including the detection of persistent epigenetic modifications, the specific detection of CRISPR-Cas complexes, a promising tool for achieving CRISPR resistance in phage therapy, the restriction of editing activity to specific tissues or developmental stages, the enhancement of microbial gene-editing strategies, and the mitigation of toxic effects of genome editing.

Genome editing is currently being performed on more than 40 crop species across 25 countries to improve variety of traits, including agronomy, the quality of food and feed, and tolerance to abiotic stress in plants. There are currently six genome-edited agricultural traits available for commercialization; these traits may be found in soybeans, canola, rice, maize, mushrooms, and camelina (Menz et al., 2020; Pixley et al., 2022). There are numerous possible risks associated with genome-edited crops such as non-target mutations, breaking of natural reproductive barriers and intermediate transgenic elements (Pixley et al., 2022). The revolutionary discovery of Acr proteins has given us greater control over CRISPR-Cas editing (Pawluk et al., 2016a). There are a variety of potential applications for ACRs in the field of plant genome editing that can be tuned, such as (i) integration of cell-specific miRNA binding sites into ACRs to construct a cell type–specific Cas9-ON switch to alter plant genomes in a cell type–specific manner (Hoffmann et al., 2019) and (ii) enabling inducible plant genome editing by fusing a light-responsive domain with the CRISPR-Cas system to enable optogenetic control of the system (Bubeck et al., 2018; Liu et al., 2022). For instance, CASANOVA, which stands for “CRISPR-Cas9 activity switching via a novel optogenetic variant of AcrIIA4”, is a chimeric protein composed of AcrIIA4 fused to the LOV2 photosensor domain from the Avena sativa phototropin-1 protein. This domain is responsible for the protein’s ability to detect light (Yu and Marchisio, 2020). Acr can help with selective breeding, which could help plants and animals improve their valuable features. The Acr can be utilized as a helpful “off-switch” for the production of Cas9 activity in the gene therapy technique, as we know that phages are employed for gene therapy for the treatment of bacterial illness (Pawluk et al., 2016a). The type I-F Acr gene family has been shown to inhibit the type I-F CRISPR system in both Pseudomonas aeruginosa and Pectobacterium atrosepticum, and type II-A Acr proteins can block the expression of Cas9 protein in most studies (Marino et al., 2018; Mir et al., 2018; Bhoobalan-Chitty et al., 2019; Athukoralage et al., 2020). Acr protein not only inhibits some Cas proteins during the editing process but also allows us to fix mistakes or off-target mutations afterward while also partially blocking editing at the targeted location (Athukoralage et al., 2020). Numerous studies have shown that Acr proteins have a wide spectrum of functional activity, allowing researchers to use them to modify the insertion, deletion, silence, and single-letter fixation of any characteristic. Identifying the most prevalent architectural or dynamic aspects of Acr-Cas interactions is crucial for predicting inhibitory consequences with novel or planned Acrs; hence, studying these biophysical principles is of paramount importance. Because of their high affinity and specificity for CRISPR-Cas systems, Acrs have the potential to cure a wide range of diseases, including those caused by multidrug-resistant bacteria; secondary bacterial infections associated with COVID-19 and SARS-CoV; disorders associated with defective genes like Alzheimer, Parkinson, and Huntington’s diseases; diseases transmitted by insects; and viral diseases through regulated genome editing. At this point of time, the Acr protein–based treatments and the role that they play in plant research are in its nascent stage. Exploring the biophysical principles important for Acr function is essential to pinpointing the most common architectural or dynamic features of Acr-Cas interactions, which can be used to predict future inhibitory outcomes with novel or designed Acrs.

NC, DT, RV, and B-HJ designed the idea and write the first draft of the manuscript; VY, KY, RG, ND, MHA, MSA, and TG reviewed the manuscript; MC, DT, LE, AG, and NC prepared the final draft; RV and B-HJ finalized and submitted the manuscript. All authors contributed to the article and approved the submitted version.