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Bacteria use a variety of defense systems to protect themselves from phage infection. In turn, phages have evolved diverse counter-defense measures to overcome host defenses. Here, we use protein structural similarity and gene co-occurrence analyses to screen >66 million viral protein sequences and >330,000 metagenome-assembled genomes for the identification of anti-phage and counter-defense systems. We predict structures for ~300,000 proteins and perform large-scale, pairwise comparison to known anti-CRISPR (Acr) and anti-phage proteins to identify structural homologs that otherwise may not be uncovered using primary sequence search. This way, we identify a Bacteroidota phage Acr protein that inhibits Cas12a, and an Akkermansia muciniphila anti-phage defense protein, termed BxaP. Gene bxaP is found in loci encoding Bacteriophage Exclusion (BREX) and restriction-modification defense systems, but confers immunity independently. Our work highlights the advantage of combining protein structural features and gene co-localization information in studying host-phage interactions. Bacteria use various defense systems to protect themselves from phage infection, and phages have evolved diverse counter-defense measures to overcome host defenses. Here, the authors use protein structural similarity and gene co-occurrence analyses for identification of new anti-phage and counter-defense systems.

The development of precise and controlled CRISPR-Cas tools has been made possible by the discovery of protein inhibitors of CRISPR-Cas systems, called anti-CRISPRs (Acrs). The Acr protein has the ability to control off-targeted mutations and impede Cas protein–editing operations. Acr can help with selective breeding, which could help plants and animals improve their valuable features. In this review, the Acr protein–based inhibitory mechanisms that have been adopted by several Acrs, such as (a) the interruption of CRISPR-Cas complex assembly, (b) interference with target DNA binding, (c) blocking of target DNA/RNA cleavage, and (d) enzymatic modification or degradation of signalling molecules, were discussed. In addition, this review emphasizes the applications of Acr proteins in the plant research.

Prokaryotes possess CRISPR-Cas systems to exclude parasitic predators, such as phages and mobile genetic elements (MGEs). These predators, in turn, encode anti-CRISPR (Acr) proteins to evade the CRISPR-Cas immunity. Recently, AcrVA4, an Acr protein inhibiting the CRISPR-Cas12a system, was shown to diminish Lachnospiraceae bacterium Cas12a (LbCas12a)-mediated genome editing in human cells, but the underlying mechanisms remain elusive. Here we report the cryo-EM structures of AcrVA4 bound to CRISPR RNA (crRNA)-loaded LbCas12a and found AcrVA4 could inhibit LbCas12a at several stages of the CRISPR-Cas working pathway, different from other characterized type I/II Acr inhibitors which target only 1 stage. First, it locks the conformation of the LbCas12a-crRNA complex to prevent target DNA-crRNA hybridization. Second, it interacts with the LbCas12a-crRNA-dsDNA complex to release the bound DNA before cleavage. Third, AcrVA4 binds the postcleavage LbCas12a complex to possibly block enzyme recycling. These findings highlight the multifunctionality of AcrVA4 and provide clues for developing regulatory genome-editing tools.

Anti-CRISPR (Acr) proteins are encoded by phages to inactivate CRISPR–Cas systems of bacteria and archaea and are used to enhance the CRISPR toolbox for genome editing. Here we report the structure and mechanism of AcrIF24, an Acr protein that inhibits the type I-F CRISPR–Cas system from Pseudomonas aeruginosa. AcrIF24 is a homodimer that associates with two copies of the surveillance complex (Csy) and prevents the hybridization between CRISPR RNA and target DNA. Furthermore, AcrIF24 functions as an anti-CRISPR-associated (Aca) protein to repress the transcription of the acrIF23-acrIF24 operon. Alone or in complex with Csy, AcrIF24 is capable of binding to the acrIF23-acrIF24 promoter DNA with nanomolar affinity. The structure of a Csy–AcrIF24–promoter DNA complex at 2.7 Å reveals the mechanism for transcriptional suppression. Our results reveal that AcrIF24 functions as an Acr-Aca fusion protein, and they extend understanding of the diverse mechanisms used by Acr proteins.Mukherjee et al. report that AcrIF24 is an Acr-Aca fusion protein that inhibits the Csy complex and suppresses transcription from the acrIF23–acrIF24 promoter, and they present cryo-EM structures to reveal the mechanism for both roles of AcrIF24.

Prime editing (PE) enables the precise installation of intended base substitutions, small deletions or small insertions into the genome of living cells. While the use of Cas9 nickase can avoid DNA double-strand breaks (DSB), undesired insertions and deletions (indels) often accompany the correct edits, particularly when PE activity increased. Here we show that the anti-CRISPR (Acr) protein AcrIIA5 can significantly enhance PE activity by up to 8.2-fold while markedly reducing byproduct indels. Further investigation reveals that AcrIIA5 can promote PE across various approaches (PE2, PE3, PE4, PE5, and PE6), edit types (substitutions, insertions and deletions), and endogenous loci. Mechanistically, AcrIIA5 appears to inhibit the re-nicking activity of PE complex rather than enhancing the core editing machinery itself, suggesting a distinct mode of interaction with Cas9. Overall, we demonstrate that a known “inhibitor” Acr protein can unexpectedly acting as an “enhancer” of CRISPR/Cas-based genome editing, providing an effective strategy to optimize PE specificity and activity. Prime editing enables precise genetic modification but often suffers from unwanted byproducts. Here, authors show that the anti-CRISPR protein AcrIIA5 unexpectedly enhances prime editing efficiency while reducing unintended indels, offering an effective strategy to improve activity and specificity.

This study reports the identification of an archaeal virus Acr protein that blocks the dissociation of the target RNA from the type III-B CRISPR–Cas effector complex, which prevents the recycling of the complex.

CRISPR–Cas adaptive immune systems protect bacteria and archaea against their invading genetic parasites, including bacteriophages/viruses and plasmids. In response to this immunity, many phages have anti-CRISPR (Acr) proteins that inhibit CRISPR–Cas targeting. To date, anti-CRISPR genes have primarily been discovered in phage or prophage genomes. Here, we uncovered acr loci on plasmids and other conjugative elements present in Firmicutes using the Listeria acrIIA1 gene as a marker. The four identified genes, found in Listeria, Enterococcus, Streptococcus and Staphylococcus genomes, can inhibit type II-A SpyCas9 or SauCas9, and are thus named acrIIA16– 19 . In Enterococcus faecalis , conjugation of a Cas9-targeted plasmid was enhanced by anti-CRISPRs derived from Enterococcus conjugative elements, highlighting a role for Acrs in the dissemination of plasmids. Reciprocal co-immunoprecipitation showed that each Acr protein interacts with Cas9, and Cas9–Acr complexes were unable to cleave DNA. Northern blotting suggests that these anti-CRISPRs manipulate single guide RNA length, loading or stability. Mirroring their activity in bacteria, AcrIIA16 and AcrIIA17 provide robust and highly potent broad-spectrum inhibition of distinct Cas9 proteins in human cells (for example, SpyCas9, SauCas9, SthCas9, NmeCas9 and CjeCas9). This work presents a focused analysis of non-phage Acr proteins, demonstrating a role in horizontal gene transfer bolstered by broad-spectrum CRISPR–Cas9 inhibition. A search for genes encoding anti-CRISPR (Acr) proteins on plasmids and other conjugative elements identifies new Acrs that regulate plasmid dissemination and are broad-spectrum inhibitors of Cas9 in human cells.

Amino acid metabolic enzymes often contain a regulatory ACT domain, named for aspartate kinase, chorismate mutase, and TyrA (prephenate dehydrogenase). Arabidopsis encodes 12 putative amino acid sensor ACT repeat (ACR) proteins, all containing ACT repeats but no identifiable catalytic domain. Arabidopsis ACRs comprise three groups based on domain composition and sequence: group I and II ACRs contain four ACTs each, and group III ACRs contain two ACTs. Previously, all three groups had been documented only in Arabidopsis. Here, we extended this to algae and land plants, showing that all three groups of ACRs are present in most, if not all, land plants, whereas among algal ACRs, although quite diverse, only group III is conserved. The appearance of canonical group I and II ACRs thus accompanied the evolution of plants from living in water to living on land. Alignment of ACTs from plant ACRs revealed a conserved motif, DRPGLL, at the putative ligand-binding site. Notably, the unique features of the DRPGLL motifs in each ACT domain are conserved in ACRs from algae to land plants. The conservation of plant ACRs is reminiscent of that of human cellular arginine sensor for mTORC1 (CASTOR1), a member of a small protein family highly conserved in animals. CASTOR proteins also have four ACT domains, although the sequence identities between ACRs and CASTORs are very low. Thus, plant ACRs and animal CASTORs may have adapted the regulatory ACT domains from a more ancient metabolic enzyme, and then evolved independently.

CRISPR-Cas systems provide prokaryotic hosts with adaptive immunity against mobile genetic elements. Many bacteriophages encode anti-CRISPR (Acr) proteins that inhibit host defense. The identification of Acr proteins is challenging due to their small size and high sequence diversity, and only a limited number has been characterized to date. In this study, we report the discovery of a novel Acr protein, AcrIB2, encoded by the φCD38-2 Clostridioides difficile phage that efficiently inhibits interference by the type I-B CRISPR-Cas system of the host and likely acts as a DNA mimic. Most C. difficile strains contain two cas operons, one encoding a full set of interference and adaptation proteins and another encoding interference proteins only. Unexpectedly, we demonstrate that only the partial operon is required for interference and is subject to inhibition by AcrIB2. Clostridioides difficile is the widespread anaerobic spore-forming bacterium that is a major cause of potentially lethal nosocomial infections associated with antibiotic therapy worldwide. Due to the increase in severe forms associated with a strong inflammatory response and higher recurrence rates, a current imperative is to develop synergistic and alternative treatments for C. difficile infections. In particular, phage therapy is regarded as a potential substitute for existing antimicrobial treatments. However, it faces challenges because C. difficile has highly active CRISPR-Cas immunity, which may be a specific adaptation to phage-rich and highly crowded gut environment. To overcome this defense, C. difficile phages must employ anti-CRISPR mechanisms. Here, we present the first anti-CRISPR protein that inhibits the CRISPR-Cas defense system in this pathogen. Our work offers insights into the interactions between C. difficile and its phages, paving the way for future CRISPR-based applications and development of effective phage therapy strategies combined with the engineering of virulent C. difficile infecting phages.

The ongoing evolutionary competition between bacteria and their phage parasites has led to the development of intricate systems of attacks, defenses, and counter-defenses. Among the prokaryotic adaptive immunity measures, the CRISPR–Cas system has garnered significant attention for its versatility in genome editing and other biotechnological applications. In this system, bacteria capture specific sequences from invading phages and store them in their genome as a record. This allows the bacteria to recognize and target the phage DNA for degradation using Cas proteins upon subsequent infections. On the phage side of the battle, a counter mechanism known as ‘Anti-CRISPR’ (Acr) has emerged. Acr proteins, encoded by phages or Mobile Genetic Elements (MGEs), function to inhibit CRISPR–Cas activity. They achieve this by either blocking DNA binding or inhibiting the cleavage process of the CRISPR–Cas effector complexes. Acrs present the potential to develop regulated and reversible CRISPR–Cas-mediated gene circuits, opening up possibilities for precise genome editing. Researchers are exploring the applications of Acrs to reduce off-target mutations associated with CRISPR–Cas, modulate the activities of Cas endonucleases, and create “genome safeguard” systems where further editing is restricted. The study and utilization of Acrs hold promise for advancing the precision and control of CRISPR–Cas technologies. Graphical abstract Enhancing CRISPR with Anti-CRISPR Protein