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.     

Mechanistic insights into the inhibition of the CRISPR-Cas surveillance complex by anti-CRISPR protein AcrIF13

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins provide prokaryotes with nucleic acid–based adaptive immunity against infections of mobile genetic elements, including phages. To counteract this immune process, phages have evolved various anti-CRISPR (Acr) proteins which deactivate CRISPR-Cas–based immunity. However, the mechanisms of many of these Acr-mediated inhibitions are not clear. Here, we report the crystal structure of AcrIF13 and explore its inhibition mechanism. The structure of AcrIF13 is unique and displays a negatively charged surface. Additionally, biochemical studies identified that AcrIF13 interacts with the type I-F CRISPR-Cas surveillance complex (Csy complex) to block target DNA recognition and that the Cas5f-8f tail and Cas7.6f subunit of the Csy complex are specific binding targets of AcrIF13. Further mutational studies demonstrated that several negatively charged residues of AcrIF13 and positively charged residues of Cas8f and Cas7f of the Csy complex are involved in AcrIF13–Csy binding. Together, our findings provide mechanistic insights into the inhibition mechanism of AcrIF13 and further suggest the prevalence of the function of Acr proteins as DNA mimics.     

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.     

Structural basis for inhibition of the type I-F CRISPR–Cas surveillance complex by AcrIF4, AcrIF7 and AcrIF14

CRISPR–Cas systems are adaptive immune systems in bacteria and archaea to defend against mobile genetic elements (MGEs) and have been repurposed as genome editing tools. Anti-CRISPR (Acr) proteins are produced by MGEs to counteract CRISPR–Cas systems and can be used to regulate genome editing by CRISPR techniques. Here, we report the cryo-EM structures of three type I-F Acr proteins, AcrIF4, AcrIF7 and AcrIF14, bound to the type I-F CRISPR–Cas surveillance complex (the Csy complex) from Pseudomonas aeruginosa. AcrIF4 binds to an unprecedented site on the C-terminal helical bundle of Cas8f subunit, precluding conformational changes required for activation of the Csy complex. AcrIF7 mimics the PAM duplex of target DNA and is bound to the N-terminal DNA vise of Cas8f. Two copies of AcrIF14 bind to the thumb domains of Cas7.4f and Cas7.6f, preventing hybridization between target DNA and the crRNA. Our results reveal structural detail of three AcrIF proteins, each binding to a different site on the Csy complex for inhibiting degradation of MGEs.     

Mobile element warfare via CRISPR and anti-CRISPR in Pseudomonas aeruginosa

Bacteria deploy multiple defenses to prevent mobile genetic element (MGEs) invasion. CRISPR–Cas immune systems use RNA-guided nucleases to target MGEs, which counter with anti-CRISPR (Acr) proteins. Our understanding of the biology and co-evolutionary dynamics of the common Type I-C CRISPR–Cas subtype has lagged because it lacks an in vivo phage-host model system. Here, we show the anti-phage function of a Pseudomonas aeruginosa Type I-C CRISPR–Cas system encoded on a conjugative pKLC102 island, and its Acr-mediated inhibition by distinct MGEs. Seven genes with anti-Type I-C function (acrIC genes) were identified, many with highly acidic amino acid content, including previously described DNA mimic AcrIF2. Four of the acr genes were broad spectrum, also inhibiting I-E or I-F P. aeruginosa CRISPR–Cas subtypes. Dual inhibition comes at a cost, however, as simultaneous expression of Type I-C and I-F systems renders phages expressing the dual inhibitor AcrIF2 more sensitive to targeting. Mutagenesis of numerous acidic residues in AcrIF2 did not impair anti-I-C or anti-I-F function per se but did exacerbate inhibition defects during competition, suggesting that excess negative charge may buffer DNA mimics against competition. Like AcrIF2, five of the Acr proteins block Cascade from binding DNA, while two function downstream, likely preventing Cas3 recruitment or activity. One such inhibitor, AcrIC3, is found in an ‘anti-Cas3’ cluster within conjugative elements, encoded alongside bona fide Cas3 inhibitors AcrIF3 and AcrIE1. Our findings demonstrate an active battle between an MGE-encoded CRISPR–Cas system and its diverse MGE targets.     

Insights into the dual functions of AcrIF14 during the inhibition of type I-F CRISPR–Cas surveillance complex

CRISPR–Cas systems are bacterial adaptive immune systems, and phages counteract these systems using many approaches such as producing anti-CRISPR (Acr) proteins. Here, we report the structures of both AcrIF14 and its complex with the crRNA-guided surveillance (Csy) complex. Our study demonstrates that apart from interacting with the Csy complex to block the hybridization of target DNA to the crRNA, AcrIF14 also endows the Csy complex with the ability to interact with non-sequence-specific dsDNA as AcrIF9 does. Further structural studies of the Csy–AcrIF14–dsDNA complex and biochemical studies uncover that the PAM recognition loop of the Cas8f subunit of the Csy complex and electropositive patches within the N-terminal domain of AcrIF14 are essential for the non-sequence-specific dsDNA binding to the Csy–AcrIF14 complex, which is different from the mechanism of AcrIF9. Our findings highlight the prevalence of Acr-induced non-specific DNA binding and shed light on future studies into the mechanisms of such Acr proteins.     

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.     

The novel anti-CRISPR AcrIIA22 relieves DNA torsion in target plasmids and impairs SpyCas9 activity

To overcome CRISPR-Cas defense systems, many phages and mobile genetic elements (MGEs) encode CRISPR-Cas inhibitors called anti-CRISPRs (Acrs). Nearly all characterized Acrs directly bind Cas proteins to inactivate CRISPR immunity. Here, using functional metagenomic selection, we describe AcrIIA22, an unconventional Acr found in hypervariable genomic regions of clostridial bacteria and their prophages from human gut microbiomes. AcrIIA22 does not bind strongly to SpyCas9 but nonetheless potently inhibits its activity against plasmids. To gain insight into its mechanism, we obtained an X-ray crystal structure of AcrIIA22, which revealed homology to PC4-like nucleic acid–binding proteins. Based on mutational analyses and functional assays, we deduced that acrIIA22 encodes a DNA nickase that relieves torsional stress in supercoiled plasmids. This may render them less susceptible to SpyCas9, which uses free energy from negative supercoils to form stable R-loops. Modifying DNA topology may provide an additional route to CRISPR-Cas resistance in phages and MGEs.

Derived from phages of the gut microbiome, this study describes a CRISPR-Cas9 inhibitor with an unexpected mechanism; instead of binding Cas9 itself, this “anti-CRISPR” relaxes plasmid DNA and enables Cas9 evasion, suggesting that DNA topology is an underappreciated battleground in phage-bacterial conflicts.     

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.     

In Silico Approaches for Prediction of Anti-CRISPR Proteins

Numerous viruses infecting bacteria and archaea encode CRISPR-Cas system inhibitors, known as anti-CRISPR proteins (Acr). The Acrs typically are highly specific for particular CRISPR variants, resulting in remarkable sequence and structural diversity and complicating accurate prediction and identification of Acrs. In addition to their intrinsic interest for understanding the coevolution of defense and counter-defense systems in prokaryotes, Acrs could be natural, potent on–off switches for CRISPR-based biotechnological tools, so their discovery, characterization and application are of major importance. Here we discuss the computational approaches for Acr prediction. Due to the enormous diversity and likely multiple origins of the Acrs, sequence similarity searches are of limited use. However, multiple features of protein and gene organization have been successfully harnessed to this end including small protein size and distinct amino acid compositions of the Acrs, association of acr genes in virus genomes with genes encoding helix-turn-helix proteins that regulate Acr expression (Acr-associated proteins, Aca), and presence of self-targeting CRISPR spacers in bacterial and archaeal genomes containing Acr-encoding proviruses. Productive approaches for Acr prediction also involve genome comparison of closely related viruses, of which one is resistant and the other one is sensitive to a particular CRISPR variant, and “guilt by association” whereby genes adjacent to a homolog of a known Aca are identified as candidate Acrs. The distinctive features of Acrs are employed for Acr prediction both by developing dedicated search algorithms and through machine learning. New approaches will be needed to identify novel types of Acrs that are likely to exist.     

Regulation of the Type I-F CRISPR-Cas system by CRP-cAMP and GalM controls spacer acquisition and interference

The CRISPR-Cas prokaryotic ‘adaptive immune systems’ represent a sophisticated defence strategy providing bacteria and archaea with protection from invading genetic elements, such as bacteriophages or plasmids. Despite intensive research into their mechanism and application, how CRISPR-Cas systems are regulated is less clear, and nothing is known about the regulation of Type I-F systems. We used Pectobacterium atrosepticum, a Gram-negative phytopathogen, to study CRISPR-Cas regulation, since it contains a single Type I-F system. The CRP-cAMP complex activated the cas operon, increasing the expression of the adaptation genes cas1 and cas2–3 in addition to the genes encoding the Csy surveillance complex. Mutation of crp or cyaA (encoding adenylate cyclase) resulted in reductions in both primed spacer acquisition and interference. Furthermore, we identified a galactose mutarotase, GalM, which reduced cas operon expression in a CRP- and CyaA-dependent manner. We propose that the Type I-F system senses metabolic changes, such as sugar availability, and regulates cas genes to initiate an appropriate defence response. Indeed, elevated glucose levels reduced cas expression in a CRP- and CyaA-dependent manner. Taken together, these findings highlight that a metabolite-sensing regulatory pathway controls expression of the Type I-F CRISPR-Cas system to modulate levels of adaptation and interference.     

Anti-CRISPR蛋白AcrVA2的结构生物学研究

大多数古生菌及半数细菌都含有成簇有规律间隔的短回文重复序列(clustered regularly interspaced short palindromic repeats,CRISPR)和CRISPR相关(CRISPR-associated,Cas)蛋白质构成的适应性免疫系统,来抵御外界噬菌体的入侵.而噬菌体为了对抗这种免疫系统,也进化出许多抗CRISPR (anti-CRISPR,Acr)的蛋白质,使得CRISPR-Cas系统受到抑制.来自牛眼莫拉氏菌(Moraxella bovoculi)的AcrVA2是目前发现的可抑制V-A型CRISPR-Cas系统效应蛋白Cas12a发挥切割活性的Acr蛋白之一,其作用机理尚不清楚.本文解析了自由状态的AcrVA2和MbCas12a^620-636-AcrVA2复合物的晶体结构,发现AcrVA2蛋白采用了一种新的α-β折叠结构,且只与自由状态的Cas12a结合.此外,AcrVA2与MbCas12a^620-636的结合主要依靠氢键和盐桥的相互作用力,并通过疏水界面得到进一步稳定.这些结果提示,AcrV...     

Functional characterization of diverse type I-F CRISPR-associated transposons

CRISPR-Cas systems generally provide adaptive immunity in prokaryotes through RNA-guided degradation of foreign genetic elements like bacteriophages and plasmids. Recently, however, transposon-encoded and nuclease-deficient CRISPR-Cas systems were characterized and shown to be co-opted by Tn7-like transposons for CRISPR RNA-guided DNA transposition. As a genome engineering tool, these CRISPR-Cas systems and their associated transposon proteins can be deployed for programmable, site-specific integration of sizable cargo DNA, circumventing the need for DNA cleavage and homology-directed repair involving endogenous repair machinery. Here, we selected a diverse set of type I-F3 CRISPR-associated transposon systems derived from Gammaproteobacteria, predicted all components essential for transposition activity, and deployed them for functionality testing within Escherichia coli. Our results demonstrate that these systems possess a significant range of integration efficiencies with regards to temperature, transposon size, and flexible PAM requirements. Additionally, our findings support the categorization of these systems into functional compatibility groups for efficient and orthogonal RNA-guided DNA integration. This work expands the CRISPR-based toolbox with new CRISPR RNA-guided DNA integrases that can be applied to complex and extensive genome engineering efforts.     

The Susceptibility of Pseudomonas aeruginosa Strains from Cystic Fibrosis Patients to Bacteriophages

Phage therapy may become a complement to antibiotics in the treatment of chronic Pseudomonas aeruginosa infection. To design efficient therapeutic cocktails, the genetic diversity of the species and the spectrum of susceptibility to bacteriophages must be investigated. Bacterial strains showing high levels of phage resistance need to be identified in order to decipher the underlying mechanisms. Here we have selected genetically diverse P. aeruginosa strains from cystic fibrosis patients and tested their susceptibility to a large collection of phages. Based on plaque morphology and restriction profiles, six different phages were purified from “pyophage”, a commercial cocktail directed against five different bacterial species, including P. aeruginosa. Characterization of these phages by electron microscopy and sequencing of genome fragments showed that they belong to 4 different genera. Among 47 P. aeruginosa strains, 13 were not lysed by any of the isolated phages individually or by pyophage. We isolated two new phages that could lyse some of these strains, and their genomes were sequenced. The presence/absence of a CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeats and Crisper associated genes) was investigated to evaluate the role of the system in phage resistance. Altogether, the results show that some P. aeruginosa strains cannot support the growth of any of the tested phages belonging to 5 different genera, and suggest that the CRISPR-Cas system is not a major defence mechanism against these lytic phages.     

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.     

Priming in the Type I-F CRISPR-Cas system triggers strand-independent spacer acquisition,bi-directionally from the primed protospacer

Clustered regularly interspaced short palindromic repeats (CRISPR), in combination with CRISPR associated (cas) genes, constitute CRISPR-Cas bacterial adaptive immune systems. To generate immunity, these systems acquire short sequences of nucleic acids from foreign invaders and incorporate these into their CRISPR arrays as spacers. This adaptation process is the least characterized step in CRISPR-Cas immunity. Here, we used Pectobacterium atrosepticum to investigate adaptation in Type I-F CRISPR-Cas systems. Pre-existing spacers that matched plasmids stimulated hyperactive primed acquisition and resulted in the incorporation of up to nine new spacers across all three native CRISPR arrays. Endogenous expression of the cas genes was sufficient, yet required, for priming. The new spacers inhibited conjugation and transformation, and interference was enhanced with increasing numbers of new spacers. We analyzed ∼350 new spacers acquired in priming events and identified a 5′-protospacer-GG-3′ protospacer adjacent motif. In contrast to priming in Type I-E systems, new spacers matched either plasmid strand and a biased distribution, including clustering near the primed protospacer, suggested a bi-directional translocation model for the Cas1:Cas2–3 adaptation machinery. Taken together these results indicate priming adaptation occurs in different CRISPR-Cas systems, that it can be highly active in wild-type strains and that the underlying mechanisms vary.