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1. AcrFinder: genome mining anti-CRISPR operons in prokaryotes and their viruses

   https://doi.org/10.1093/nar/gkaa351  

   Yi, Haidong ; Huang, Le ; Yang, Bowen ; Gomez, Javi ; Zhang, Han ; Yin, Yanbin ( May 2020 , Nucleic Acids Research)

   Abstract Anti-CRISPR (Acr) proteins encoded by (pro)phages/(pro)viruses have a great potential to enable a more controllable genome editing. However, genome mining new Acr proteins is challenging due to the lack of a conserved functional domain and the low sequence similarity among experimentally characterized Acr proteins. We introduce here AcrFinder, a web server (http://bcb.unl.edu/AcrFinder) that combines three well-accepted ideas used by previous experimental studies to pre-screen genomic data for Acr candidates. These ideas include homology search, guilt-by-association (GBA), and CRISPR-Cas self-targeting spacers. Compared to existing bioinformatics tools, AcrFinder has the following unique functions: (i) it is the first online server specifically mining genomes for Acr-Aca operons; (ii) it provides a most comprehensive Acr and Aca (Acr-associated regulator) database (populated by GBA-based Acr and Aca datasets); (iii) it combines homology-based, GBA-based, and self-targeting approaches in one software package; and (iv) it provides a user-friendly web interface to take both nucleotide and protein sequence files as inputs, and output a result page with graphic representation of the genomic contexts of Acr-Aca operons. The leave-one-out cross-validation on experimentally characterized Acr-Aca operons showed that AcrFinder had a 100% recall. AcrFinder will be a valuable web resource to help experimental microbiologists discover new Anti-CRISPRs. 

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2. Bioinformatics Identification of Anti-CRISPR Loci by Using Homology, Guilt-by-Association, and CRISPR Self-Targeting Spacer Approaches

   https://doi.org/10.1128/mSystems.00455-19  

   Yin, Yanbin ; Yang, Bowen ; Entwistle, Sarah ; Claesson, Marcus J. ( October 2019 , mSystems)

   ABSTRACT Anti-CRISPR (Acr) loci/operons encode Acr proteins and Acr-associated (Aca) proteins. Forty-five Acr families have been experimentally characterized inhibiting seven subtypes of CRISPR-Cas systems. We have developed a bioinformatics pipeline to identify genomic loci containing Acr homologs and/or Aca homologs by combining three computational approaches: homology, guilt-by-association, and self-targeting spacers. Homology search found thousands of Acr homologs in bacterial and viral genomes, but most are homologous to AcrIIA7 and AcrIIA9. Investigating the gene neighborhood of these Acr homologs revealed that only a small percentage (23.0% in bacteria and 8.2% in viruses) of them have neighboring Aca homologs and thus form Acr-Aca operons. Surprisingly, although a self-targeting spacer is a strong indicator of the presence of Acr genes in a genome, a large percentage of Acr-Aca loci are found in bacterial genomes without self-targeting spacers or even without complete CRISPR-Cas systems. Additionally, for Acr homologs from genomes with self-targeting spacers, homology-based Acr family assignments do not always agree with the self-targeting CRISPR-Cas subtypes. Last, by investigating Acr genomic loci coexisting with self-targeting spacers in the same genomes, five known subtypes (I-C, I-E, I-F, II-A, and II-C) and five new subtypes (I-B, III-A, III-B, IV-A, and V-U4) of Acrs were inferred. Based on these findings, we conclude that the discovery of new anti-CRISPRs should not be restricted to genomes with self-targeting spacers and loci with Acr homologs. The evolutionary arms race of CRISPR-Cas systems and anti-CRISPR systems may have driven the adaptive and rapid gain and loss of these elements in closely related genomes. IMPORTANCE As a naturally occurring adaptive immune system, CRISPR-Cas (clustered regularly interspersed short palindromic repeats–CRISPR-associated genes) systems are widely found in bacteria and archaea to defend against viruses. Since 2013, the application of various bacterial CRISPR-Cas systems has become very popular due to their development into targeted and programmable genome engineering tools with the ability to edit almost any genome. As the natural off-switch of CRISPR-Cas systems, anti-CRISPRs have a great potential to serve as regulators of CRISPR-Cas tools and enable safer and more controllable genome editing. This study will help understand the relative usefulness of the three bioinformatics approaches for new Acr discovery, as well as guide the future development of new bioinformatics tools to facilitate anti-CRISPR research. The thousands of Acr homologs and hundreds of new anti-CRISPR loci identified in this study will be a valuable data resource for genome engineers to search for new CRISPR-Cas regulators. 

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3. Inhibition mechanisms of AcrF9, AcrF8, and AcrF6 against type I-F CRISPR–Cas complex revealed by cryo-EM

   https://doi.org/10.1073/pnas.1922638117  

   Zhang, Kaiming ; Wang, Shuo ; Li, Shanshan ; Zhu, Yuwei ; Pintilie, Grigore D. ; Mou, Tung-Chung ; Schmid, Michael F. ; Huang, Zhiwei ; Chiu, Wah ( March 2020 , Proceedings of the National Academy of Sciences)

   null (Ed.)

   Prokaryotes and viruses have fought a long battle against each other. Prokaryotes use CRISPR–Cas-mediated adaptive immunity, while conversely, viruses evolve multiple anti-CRISPR (Acr) proteins to defeat these CRISPR–Cas systems. The type I-F CRISPR–Cas system in Pseudomonas aeruginosa requires the crRNA-guided surveillance complex (Csy complex) to recognize the invading DNA. Although some Acr proteins against the Csy complex have been reported, other relevant Acr proteins still need studies to understand their mechanisms. Here, we obtain three structures of previously unresolved Acr proteins (AcrF9, AcrF8, and AcrF6) bound to the Csy complex using electron cryo-microscopy (cryo-EM), with resolution at 2.57 Å, 3.42 Å, and 3.15 Å, respectively. The 2.57-Å structure reveals fine details for each molecular component within the Csy complex as well as the direct and water-mediated interactions between proteins and CRISPR RNA (crRNA). Our structures also show unambiguously how these Acr proteins bind differently to the Csy complex. AcrF9 binds to key DNA-binding sites on the Csy spiral backbone. AcrF6 binds at the junction between Cas7.6f and Cas8f, which is critical for DNA duplex splitting. AcrF8 binds to a distinct position on the Csy spiral backbone and forms interactions with crRNA, which has not been seen in other Acr proteins against the Csy complex. Our structure-guided mutagenesis and biochemistry experiments further support the anti-CRISPR mechanisms of these Acr proteins. Our findings support the convergent consequence of inhibiting degradation of invading DNA by these Acr proteins, albeit with different modes of interactions with the type I-F CRISPR–Cas system. 

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4. Network-Based Prediction of Novel CRISPR-Associated Genes in Metagenomes

   https://doi.org/10.1128/mSystems.00752-19  

   Weissman, Jake L. ; Johnson, Philip L. ( February 2020 , mSystems)

   Chia, Nicholas (Ed.)

   ABSTRACT A diversity of clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems provide adaptive immunity to bacteria and archaea through recording “memories” of past viral infections. Recently, many novel CRISPR-associated proteins have been discovered via computational studies, but those studies relied on biased and incomplete databases of assembled genomes. We avoided these biases and applied a network theory approach to search for novel CRISPR-associated genes by leveraging subtle ecological cooccurrence patterns identified from environmental metagenomes. We validated our method using existing annotations and discovered 32 novel CRISPR-associated gene families. These genes span a range of putative functions, with many potentially regulating the response to infection. IMPORTANCE Every branch on the tree of life, including microbial life, faces the threat of viral pathogens. Over the course of billions of years of coevolution, prokaryotes have evolved a great diversity of strategies to defend against viral infections. One of these is the CRISPR adaptive immune system, which allows microbes to “remember” past infections in order to better fight them in the future. There has been much interest among molecular biologists in CRISPR immunity because this system can be repurposed as a tool for precise genome editing. Recently, a number of comparative genomics approaches have been used to detect novel CRISPR-associated genes in databases of genomes with great success, potentially leading to the development of new genome-editing tools. Here, we developed novel methods to search for these distinct classes of genes directly in environmental samples (“metagenomes”), thus capturing a more complete picture of the natural diversity of CRISPR-associated genes. 

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5. The Many (Inter)faces of Anti-CRISPRs: Modulation of CRISPR-Cas Structure and Dynamics by Mechanistically Diverse Inhibitors

   https://doi.org/10.3390/biom13020264  

   Belato, Helen B. ; Lisi, George P. ( February 2023 , Biomolecules)

   The discovery of protein inhibitors of CRISPR-Cas systems, called anti-CRISPRs (Acrs), has enabled the development of highly controllable and precise CRISPR-Cas tools. Anti-CRISPRs share very little structural or sequential resemblance to each other or to other proteins, which raises intriguing questions regarding their modes of action. Many structure–function studies have shed light on the mechanism(s) of Acrs, which can act as orthosteric or allosteric inhibitors of CRISPR–Cas machinery, as well as enzymes that irreversibly modify CRISPR–Cas components. Only recently has the breadth of diversity of Acr structures and functions come to light, and this remains a rapidly evolving field. Here, we draw attention to a plethora of Acr mechanisms, with particular focus on how their action toward Cas proteins modulates conformation, dynamic (allosteric) signaling, nucleic acid binding, and cleavage ability.

    

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