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.     

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.     

Inhibition mechanisms of CRISPR-Cas9 by AcrIIA17 and AcrIIA18

Mobile genetic elements such as phages and plasmids have evolved anti-CRISPR proteins (Acrs) to suppress CRISPR-Cas adaptive immune systems. Recently, several phage and non-phage derived Acrs including AcrIIA17 and AcrIIA18 have been reported to inhibit Cas9 through modulation of sgRNA. Here, we show that AcrIIA17 and AcrIIA18 inactivate Cas9 through distinct mechanisms. AcrIIA17 inhibits Cas9 activity through interference with Cas9-sgRNA binary complex formation. In contrast, AcrIIA18 induces the truncation of sgRNA in a Cas9-dependent manner, generating a shortened sgRNA incapable of triggering Cas9 activity. The crystal structure of AcrIIA18, combined with mutagenesis studies, reveals a crucial role of the N-terminal β-hairpin in AcrIIA18 for sgRNA cleavage. The enzymatic inhibition mechanism of AcrIIA18 is different from those of the other reported type II Acrs. Our results add new insights into the mechanistic understanding of CRISPR-Cas9 inhibition by Acrs, and also provide valuable information in the designs of tools for conditional manipulation of CRISPR-Cas9.     

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.     

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.     

Optogenetic control of Neisseria meningitidis Cas9 genome editing using an engineered,light-switchable anti-CRISPR protein

Optogenetic control of CRISPR–Cas9 systems has significantly improved our ability to perform genome perturbations in living cells with high precision in time and space. As new Cas orthologues with advantageous properties are rapidly being discovered and engineered, the need for straightforward strategies to control their activity via exogenous stimuli persists. The Cas9 from Neisseria meningitidis (Nme) is a particularly small and target-specific Cas9 orthologue, and thus of high interest for in vivo genome editing applications. Here, we report the first optogenetic tool to control NmeCas9 activity in mammalian cells via an engineered, light-dependent anti-CRISPR (Acr) protein. Building on our previous Acr engineering work, we created hybrids between the NmeCas9 inhibitor AcrIIC3 and the LOV2 blue light sensory domain from Avena sativa. Two AcrIIC3-LOV2 hybrids from our collection potently blocked NmeCas9 activity in the dark, while permitting robust genome editing at various endogenous loci upon blue light irradiation. Structural analysis revealed that, within these hybrids, the LOV2 domain is located in striking proximity to the Cas9 binding surface. Together, our work demonstrates optogenetic regulation of a type II-C CRISPR effector and might suggest a new route for the design of optogenetic Acrs.     

Structural basis of Staphylococcus aureus Cas9 inhibition by AcrIIA14

Bacteriophages have evolved a range of anti-CRISPR proteins (Acrs) to escape the adaptive immune system of prokaryotes, therefore Acrs can be used as switches to regulate gene editing. Herein, we report the crystal structure of a quaternary complex of AcrIIA14 bound SauCas9–sgRNA–dsDNA at 2.22 Å resolution, revealing the molecular basis for AcrIIA14 recognition and inhibition. Our structural and biochemical data analysis suggest that AcrIIA14 binds to a non-conserved region of SauCas9 HNH domain that is distinctly different from AcrIIC1 and AcrIIC3, with no significant effect on sgRNA or dsDNA binding. Further, our structural data shows that the allostery of the HNH domain close to the substrate DNA is sterically prevented by AcrIIA14 binding. In addition, the binding of AcrIIA14 triggers the conformational allostery of the HNH domain and the L1 linker within the SauCas9, driving them to make new interactions with the target-guide heteroduplex, enhancing the inhibitory ability of AcrIIA14. Our research both expands the current understanding of anti-CRISPRs and provides additional culues for the rational use of the CRISPR-Cas system in genome editing and gene regulation.     

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.     

CRISPR–Cas9-assisted recombineering in Lactobacillus reuteri

Clustered regularly interspaced palindromic repeats (CRISPRs) and the CRISPR-associated (Cas) nuclease protect bacteria and archeae from foreign DNA by site-specific cleavage of incoming DNA. Type-II CRISPR–Cas systems, such as the Streptococcus pyogenes CRISPR–Cas9 system, can be adapted such that Cas9 can be guided to a user-defined site in the chromosome to introduce double-stranded breaks. Here we have developed and optimized CRISPR–Cas9 function in the lactic acid bacterium Lactobacillus reuteri ATCC PTA 6475. We established proof-of-concept showing that CRISPR–Cas9 selection combined with single-stranded DNA (ssDNA) recombineering is a realistic approach to identify at high efficiencies edited cells in a lactic acid bacterium. We show for three independent targets that subtle changes in the bacterial genome can be recovered at efficiencies ranging from 90 to 100%. By combining CRISPR–Cas9 and recombineering, we successfully applied codon saturation mutagenesis in the L. reuteri chromosome. Also, CRISPR–Cas9 selection is critical to identify low-efficiency events such as oligonucleotide-mediated chromosome deletions. This also means that CRISPR–Cas9 selection will allow identification of recombinant cells in bacteria with low recombineering efficiencies, eliminating the need for ssDNA recombineering optimization procedures. We envision that CRISPR–Cas genome editing has the potential to change the landscape of genome editing in lactic acid bacteria, and other Gram-positive bacteria.     

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.     

Expanding the plant genome editing toolbox with recently developed CRISPR–Cas systems

Since its first appearance, CRISPR–Cas9 has been developed extensively as a programmable genome-editing tool, opening a new era in plant genome engineering. However, CRISPR–Cas9 still has some drawbacks, such as limitations of the protospacer-adjacent motif (PAM) sequence, target specificity, and the large size of the cas9 gene. To combat invading bacterial phages and plasmid DNAs, bacteria and archaea have diverse and unexplored CRISPR–Cas systems, which have the potential to be developed as a useful genome editing tools. Recently, discovery and characterization of additional CRISPR–Cas systems have been reported. Among them, several CRISPR–Cas systems have been applied successfully to plant and human genome editing. For example, several groups have achieved genome editing using CRISPR–Cas type I-D and type I-E systems, which had never been applied for genome editing previously. In addition to higher specificity and recognition of different PAM sequences, recently developed CRISPR–Cas systems often provide unique characteristics that differ from well-known Cas proteins such as Cas9 and Cas12a. For example, type I CRISPR–Cas10 induces small indels and bi-directional long-range deletions ranging up to 7.2 kb in tomatoes (Solanum lycopersicum L.). Type IV CRISPR–Cas13 targets RNA, not double-strand DNA, enabling highly specific knockdown of target genes. In this article, we review the development of CRISPR–Cas systems, focusing especially on their application to plant genome engineering. Recent CRISPR–Cas tools are helping expand our plant genome engineering toolbox.

Recently discovered and characterized clustered regularly interspaced short palindromic repeats-CRISPR associated (CRISPR–Cas) systems allow additional applications to plant genome editing.     

Prophages are associated with extensive CRISPR–Cas auto-immunity

CRISPR–Cas systems require discriminating self from non-self DNA during adaptation and interference. Yet, multiple cases have been reported of bacteria containing self-targeting spacers (STS), i.e. CRISPR spacers targeting protospacers on the same genome. STS has been suggested to reflect potential auto-immunity as an unwanted side effect of CRISPR–Cas defense, or a regulatory mechanism for gene expression. Here we investigated the incidence, distribution, and evasion of STS in over 100 000 bacterial genomes. We found STS in all CRISPR–Cas types and in one fifth of all CRISPR-carrying bacteria. Notably, up to 40% of I-B and I-F CRISPR–Cas systems contained STS. We observed that STS-containing genomes almost always carry a prophage and that STS map to prophage regions in more than half of the cases. Despite carrying STS, genetic deterioration of CRISPR–Cas systems appears to be rare, suggesting a level of escape from the potentially deleterious effects of STS by other mechanisms such as anti-CRISPR proteins and CRISPR target mutations. We propose a scenario where it is common to acquire an STS against a prophage, and this may trigger more extensive STS buildup by primed spacer acquisition in type I systems, without detrimental autoimmunity effects as mechanisms of auto-immunity evasion create tolerance to STS-targeted prophages.     

Genome editing in mammalian cells using the CRISPR type I-D nuclease

Adoption of CRISPR–Cas systems, such as CRISPR–Cas9 and CRISPR–Cas12a, has revolutionized genome engineering in recent years; however, application of genome editing with CRISPR type I—the most abundant CRISPR system in bacteria—remains less developed. Type I systems, such as type I-E, and I-F, comprise the CRISPR-associated complex for antiviral defense (‘Cascade’: Cas5, Cas6, Cas7, Cas8 and the small subunit) and Cas3, which degrades the target DNA; in contrast, for the sub-type CRISPR–Cas type I-D, which lacks a typical Cas3 nuclease in its CRISPR locus, the mechanism of target DNA degradation remains unknown. Here, we found that Cas10d is a functional nuclease in the type I-D system, performing the role played by Cas3 in other CRISPR–Cas type I systems. The type I-D system can be used for targeted mutagenesis of genomic DNA in human cells, directing both bi-directional long-range deletions and short insertions/deletions. Our findings suggest the CRISPR–Cas type I-D system as a unique effector pathway in CRISPR that can be repurposed for genome engineering in eukaryotic cells.     

Anti-CRISPR Protein AcrIIC5 Inhibits CRISPR-Cas9 by Occupying the Target DNA Binding Pocket

Anti-CRISPR proteins inhibit CRISPR-Cas immune systems through diverse mechanisms. Previously, the anti-CRISPR protein AcrIIC5_Smu was shown to potently inhibit a type II-C Cas9 from Neisseria meningitidis (Nme1Cas9). In this work, we explore the mechanism of activity of the AcrIIC5 homologue from Neisseria chenwenguii (AcrIIC5_Nch) and show that it prevents Cas9 binding to target DNA. We show that AcrIIC5_Nch targets the PAM-interacting domain (PID) of Nme1Cas9 for inhibition, agreeing with previous findings for AcrIIC5_Smu, and newly establish that strong binding of the anti-CRISPR requires guide RNA be pre-loaded on Cas9. We determined the crystal structure of AcrIIC5_Nch using X-ray crystallography and identified amino acid residues that are critical for its function. Using a protein docking algorithm we show that AcrIIC5_Nch likely occupies the Cas9 DNA binding pocket, thereby inhibiting target DNA binding through a mechanism similar to that previously described for AcrIIA2 and AcrIIA4.     

Non-canonical inhibition strategies and structural basis of anti-CRISPR proteins targeting type I CRISPR-Cas systems

Mobile genetic elements (MGEs) such as bacteriophages and their host prokaryotes are trapped in an eternal battle against each other. To cope with foreign infection, bacteria and archaea have evolved multiple immune strategies, out of which CRISPR-Cas system is up to now the only discovered adaptive system in prokaryotes. Despite the fact that CRISPR-Cas system provides powerful and delicate protection against MGEs, MGEs have also evolved anti-CRISPR proteins (Acrs) to counteract the CRISPR-Cas immune defenses. To date, 46 families of Acrs targeting type I CRISPR-Cas system have been characterized, out of which structure information of 21 families have provided insights on their inhibition strategies. Here, we review the non-canonical inhibition strategies adopted by Acrs targeting type I CRISPR-Cas systems based on their structure information by incorporating the most recent advances in this field, and discuss our current understanding and future perspectives. The delicate interplay between type I CRISPR-Cas systems and their Acrs provides us with important insights into the ongoing fierce arms race between prokaryotic hosts and their predators.     

Structural basis for substrate recognition and cleavage by the dimerization-dependent CRISPR–Cas12f nuclease

Cas12f, also known as Cas14, is an exceptionally small type V-F CRISPR–Cas nuclease that is roughly half the size of comparable nucleases of this type. To reveal the mechanisms underlying substrate recognition and cleavage, we determined the cryo-EM structures of the Cas12f-sgRNA-target DNA and Cas12f-sgRNA complexes at 3.1 and 3.9 Å, respectively. An asymmetric Cas12f dimer is bound to one sgRNA for recognition and cleavage of dsDNA substrate with a T-rich PAM sequence. Despite its dimerization, Cas12f adopts a conserved activation mechanism among the type V nucleases which requires coordinated conformational changes induced by the formation of the crRNA-target DNA heteroduplex, including the close-to-open transition in the lid motif of the RuvC domain. Only one RuvC domain in the Cas12f dimer is activated by substrate recognition, and the substrate bound to the activated RuvC domain is captured in the structure. Structure-assisted truncated sgRNA, which is less than half the length of the original sgRNA, is still active for target DNA cleavage. Our results expand our understanding of the diverse type V CRISPR–Cas nucleases and facilitate potential genome editing applications using the miniature Cas12f.     

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.     

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.