Molecular basis of dual anti-CRISPR and auto-regulatory functions of AcrIF24

CRISPR-Cas systems are adaptive immune systems in bacteria and archaea that provide resistance against phages and other mobile genetic elements. To fight against CRISPR-Cas systems, phages and archaeal viruses encode anti-CRISPR (Acr) proteins that inhibit CRISPR-Cas systems. The expression of acr genes is controlled by anti-CRISPR-associated (Aca) proteins encoded within acr-aca operons. AcrIF24 is a recently identified Acr that inhibits the type I-F CRISPR-Cas system. Interestingly, AcrIF24 was predicted to be a dual-function Acr and Aca. Here, we elucidated the crystal structure of AcrIF24 from Pseudomonas aeruginosa and identified its operator sequence within the regulated acr-aca operon promoter. The structure of AcrIF24 has a novel domain composition, with wing, head and body domains. The body domain is responsible for recognition of promoter DNA for Aca regulatory activity. We also revealed that AcrIF24 directly bound to type I-F Cascade, specifically to Cas7 via its head domain as part of its Acr mechanism. Our results provide new molecular insights into the mechanism of a dual functional Acr-Aca protein.     

Ecology and evolution of phages encoding anti-CRISPR proteins

CRISPR-Cas are prokaryotic defence systems that provide protection against invasion by mobile genetic elements (MGE), including bacteriophages. MGE can overcome CRISPR-Cas defences by encoding anti-CRISPR (Acr) proteins. These proteins are produced in the early stages of the infection and inhibit the CRISPR-Cas machinery to allow phage replication. While research on Acr has mainly focused on their discovery, structure and mode of action, and their applications in biotechnology, the impact of Acr on the ecology of MGE as well as on the coevolution with their bacterial hosts only begins to be unravelled. In this review, we summarise our current understanding on the distribution of anti-CRISPR genes in MGE, the ecology of phages encoding Acr, and their coevolution with bacterial defence mechanisms. We highlight the need to use more diverse and complex experimental models to better understand the impact of anti-CRISPR in MGE-host interactions.     

Anti-CRISPR AcrIF9 functions by inducing the CRISPR–Cas complex to bind DNA non-specifically

Phages and other mobile genetic elements express anti-CRISPR proteins (Acrs) to protect their genomes from destruction by CRISPR–Cas systems. Acrs usually block the ability of CRISPR–Cas systems to bind or cleave their nucleic acid substrates. Here, we investigate an unusual Acr, AcrIF9, that induces a gain-of-function to a type I-F CRISPR–Cas (Csy) complex, causing it to bind strongly to DNA that lacks both a PAM sequence and sequence complementarity. We show that specific and non-specific dsDNA compete for the same site on the Csy:AcrIF9 complex with rapid exchange, but specific ssDNA appears to still bind through complementarity to the CRISPR RNA. Induction of non-specific DNA-binding is a shared property of diverse AcrIF9 homologues. Substitution of a conserved positively charged surface on AcrIF9 abrogated non-specific dsDNA-binding of the Csy:AcrIF9 complex, but specific dsDNA binding was maintained. AcrIF9 mutants with impaired non-specific dsDNA binding activity in vitro displayed a reduced ability to inhibit CRISPR–Cas activity in vivo. We conclude that misdirecting the CRISPR–Cas complex to bind non-specific DNA is a key component of the inhibitory mechanism of AcrIF9. This inhibitory mechanism is distinct from a previously characterized anti-CRISPR, AcrIF1, that sterically blocks DNA-binding, even though AcrIF1and AcrIF9 bind to the same site on the Csy complex.     

Structural and mechanistic insights into the inhibition of type I-F CRISPR-Cas system by anti-CRISPR protein AcrIF23

Prokaryotes evolved clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins as a kind of adaptive immune defense against mobile genetic elements including harmful phages. To counteract this defense, many mobile genetic elements in turn encode anti-CRISPR proteins (Acrs) to inactivate the CRISPR-Cas system. While multiple mechanisms of Acrs have been uncovered, it remains unknown whether other mechanisms are utilized by uncharacterized Acrs. Here, we report a novel mechanism adopted by recently identified AcrIF23. We show that AcrIF23 interacts with the Cas2/3 helicase-nuclease in the type I-F CRISPR-Cas system, similar to AcrIF3. The structure of AcrIF23 demonstrated a novel fold and structure-based mutagenesis identified a surface region of AcrIF23 involved in both Cas2/3-binding and its inhibition capacity. Unlike AcrIF3, however, we found AcrIF23 only potently inhibits the DNA cleavage activity of Cas2/3 but does not hinder the recruitment of Cas2/3 to the CRISPR RNA-guided surveillance complex (the Csy complex). Also, in contrast to AcrIF3 which hinders substrate DNA recognition by Cas2/3, we show AcrIF23 promotes DNA binding to Cas2/3. Taken together, our study identifies a novel anti-CRISPR mechanism used by AcrIF23 and highlights the diverse mechanisms adopted by Acrs.     

Discovery of potent and versatile CRISPR–Cas9 inhibitors engineered for chemically controllable genome editing

Anti-CRISPR (Acr) proteins are encoded by many mobile genetic elements (MGEs) such as phages and plasmids to combat CRISPR–Cas adaptive immune systems employed by prokaryotes, which provide powerful tools for CRISPR–Cas-based applications. Here, we discovered nine distinct type II-A anti-CRISPR (AcrIIA24–32) families from Streptococcus MGEs and found that most Acrs can potently inhibit type II-A Cas9 orthologs from Streptococcus (SpyCas9, St1Cas9 or St3Cas9) in bacterial and human cells. Among these Acrs, AcrIIA26, AcrIIA27, AcrIIA30 and AcrIIA31 are able to block Cas9 binding to DNA, while AcrIIA24 abrogates DNA cleavage by Cas9. Notably, AcrIIA25.1 and AcrIIA32.1 can inhibit both DNA binding and DNA cleavage activities of SpyCas9, exhibiting unique anti-CRISPR characteristics. Importantly, we developed several chemically inducible anti-CRISPR variants based on AcrIIA25.1 and AcrIIA32.1 by comprising hybrids of Acr protein and the 4-hydroxytamoxifen-responsive intein, which enabled post-translational control of CRISPR–Cas9-mediated genome editing in human cells. Taken together, our work expands the diversity of type II-A anti-CRISPR families and the toolbox of Acr proteins for the chemically inducible control of Cas9-based applications.     

The structure of AcrIE4-F7 reveals a common strategy for dual CRISPR inhibition by targeting PAM recognition sites

Bacteria and archaea use the CRISPR-Cas system to fend off invasions of bacteriophages and foreign plasmids. In response, bacteriophages encode anti-CRISPR (Acr) proteins that potently inhibit host Cas proteins to suppress CRISPR-mediated immunity. AcrIE4-F7, which was isolated from Pseudomonas citronellolis, is a fused form of AcrIE4 and AcrIF7 that inhibits both type I-E and type I-F CRISPR-Cas systems. Here, we determined the structure of AcrIE4-F7 and identified its Cas target proteins. The N-terminal AcrIE4 domain adopts a novel α-helical fold that targets the PAM interaction site of the type I-E Cas8e subunit. The C-terminal AcrIF7 domain exhibits an αβ fold like native AcrIF7, which disables target DNA recognition by the PAM interaction site in the type I-F Cas8f subunit. The two Acr domains are connected by a flexible linker that allows prompt docking onto their cognate Cas8 targets. Conserved negative charges in each Acr domain are required for interaction with their Cas8 targets. Our results illustrate a common mechanism by which AcrIE4-F7 inhibits divergent CRISPR-Cas types.     

anti-CRISPR的起源、发展和应用

细菌和古菌等微生物与病毒（噬菌体）之间的生存之战是一场“军备竞赛”。细菌和古菌已经进化出多种先天和适应性的免疫系统来抵御噬菌体的入侵。噬菌体则利用不同的对抗策略来躲避这些噬菌体防御机制。CRISPR-Cas （Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-Associated）系统就是细菌和古菌广泛编码的一种抵御噬菌体等外源遗传元件的获得性免疫系统,与此同时,噬菌体也进化出特异性的anti-CRISPR来抵抗CRISPR-Cas系统的免疫。本文系统综述了anti-CRISPR的发现过程、分类和作用机制,并展望了其潜在的应用。     

Interactions between mobile genetic elements: An anti-phage gene in an integrative and conjugative element protects host cells from predation by a temperate bacteriophage

Most bacterial genomes contain horizontally acquired and transmissible mobile genetic elements, including temperate bacteriophages and integrative and conjugative elements. Little is known about how these elements interact and co-evolved as parts of their host genomes. In many cases, it is not known what advantages, if any, these elements provide to their bacterial hosts. Most strains of Bacillus subtilis contain the temperate phage SPß and the integrative and conjugative element ICEBs1. Here we show that the presence of ICEBs1 in cells protects populations of B. subtilis from predation by SPß, likely providing selective pressure for the maintenance of ICEBs1 in B. subtilis. A single gene in ICEBs1 (yddK, now called spbK for SPß killing) was both necessary and sufficient for this protection. spbK inhibited production of SPß, during both activation of a lysogen and following de novo infection. We found that expression spbK, together with the SPß gene yonE constitutes an abortive infection system that leads to cell death. spbK encodes a TIR (Toll-interleukin-1 receptor)-domain protein with similarity to some plant antiviral proteins and animal innate immune signaling proteins. We postulate that many uncharacterized cargo genes in ICEs may confer selective advantage to cells by protecting against other mobile elements.     

BOF: a novel family of bacterial OB-fold proteins

Using top-of-the-line fold recognition methods, we assigned an oligonucleotide/oligosaccharide-binding (OB)-fold structure to a family of previously uncharacterized hypothetical proteins from several bacterial genomes. This novel family of bacterial OB-fold (BOF) proteins present in a number of pathogenic strains encompasses sequences of unknown function from DUF388 (in Pfam database) and COG3111. The BOF proteins can be linked evolutionarily to other members of the OB-fold nucleic acid-binding superfamily (anticodon-binding and single strand DNA-binding domains), although they probably lack nucleic acid-binding properties as implied by the analysis of the potential binding site. The presence of conserved N-terminal predicted signal peptide indicates that BOF family members localize in the periplasm where they may function to bind proteins, small molecules, or other typical OB-fold ligands. As hypothesized for the distantly related OB-fold containing bacterial enterotoxins, the loss of nucleotide-binding function and the rapid evolution of the BOF ligand-binding site may be associated with the presence of BOF proteins in mobile genetic elements and their potential role in bacterial pathogenicity.