Structural and Mechanistic Insight into CRISPR-Cas9 Inhibition by Anti-CRISPR Protein AcrIIC4Hpa.

Phages, plasmids, and other mobile genetic elements express inhibitors of CRISPR-Cas immune systems, known as anti-CRISPR proteins, to protect themselves from targeted destruction. These anti-CRISPR proteins have been shown to function through very diverse mechanisms. In this work we investigate the activity of an anti-CRISPR isolated from a prophage in Haemophilus parainfluenzae that blocks CRISPR-Cas9 DNA cleavage activity. We determine the three-dimensional crystal structure of AcrIIC4Hpa and show that it binds to the Cas9 Recognition Domain. This binding does not prevent the Cas9-anti-CRISPR complex from interacting with target DNA but does inhibit DNA cleavage. AcrIIC4Hpa likely acts by blocking the conformational changes that allow the HNH and RuvC endonuclease domains to contact the DNA sites to be nicked.

Crystal structure of the anti-CRISPR, AcrIIC4.

Clustered regularly interspaced short palindromic repeats (CRISPRs)-CRISPR-associated protein systems are bacterial and archaeal defense mechanisms against invading elements such as phages and viruses. To overcome these defense systems, phages and viruses have developed inhibitors called anti-CRISPRs (Acrs) that are capable of inhibiting the host CRISPR-Cas system via different mechanisms. Although the inhibitory mechanisms of AcrIIC1, AcrIIC2, and AcrIIC3 have been revealed, the inhibitory mechanisms of AcrIIC4 and AcrIIC5 have not been fully understood and structural data are unavailable. In this study, we elucidated the crystal structure of Type IIC anti-CRISPR protein, AcrIIC4. Our structural analysis revealed that AcrIIC4 exhibited a helical bundle fold comprising four helixes. Further biochemical and biophysical analyses showed that AcrIIC4 formed a monomer in solution, and monomeric AcrIIC4 directly interacted with Cas9 and Cas9/sgRNA complex. Discovery of the structure of AcrIIC4 and their interaction mode on Cas9 will help us elucidate the diversity in the inhibitory mechanisms of the Acr protein family.

A genetically encoded anti-CRISPR protein constrains gene drive spread and prevents population suppression.

CRISPR-based gene drives offer promising means to reduce the burden of pests and vector-borne diseases. These techniques consist of releasing genetically modified organisms carrying CRISPR-Cas nucleases designed to bias their inheritance and rapidly propagate desired modifications. Gene drives can be intended to reduce reproductive capacity of harmful insects or spread anti-pathogen effectors through wild populations, even when these confer fitness disadvantages. Technologies capable of halting the spread of gene drives may prove highly valuable in controlling, counteracting, and even reverting their effect on individual organisms as well as entire populations. Here we show engineering and testing of a genetic approach, based on the germline expression of a phage-derived anti-CRISPR protein (AcrIIA4), able to inactivate CRISPR-based gene drives and restore their inheritance to Mendelian rates in the malaria vector Anopheles gambiae. Modeling predictions and cage testing show that a single release of male mosquitoes carrying the AcrIIA4 protein can block the spread of a highly effective suppressive gene drive preventing population collapse of caged malaria mosquitoes.

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.

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.

Development of aptamer-based inhibitors for CRISPR/Cas system.

The occurrence of accidental mutations or deletions caused by genome editing with CRISPR/Cas9 system remains a critical unsolved problem of the technology. Blocking excess or prolonged Cas9 activity in cells is considered as one means of solving this problem. Here, we report the development of an inhibitory DNA aptamer against Cas9 by means of in vitro selection (systematic evolution of ligands by exponential enrichment) and subsequent screening with an in vitro cleavage assay. The inhibitory aptamer could bind to Cas9 at low nanomolar affinity and partially form a duplex with CRISPR RNA, contributing to its inhibitory activity. We also demonstrated that improving the inhibitory aptamer with locked nucleic acids efficiently suppressed Cas9-directed genome editing in cells and reduced off-target genome editing. The findings presented here might enable the development of safer and controllable genome editing for biomedical research and gene therapy.

ZIF-C for targeted RNA interference and CRISPR/Cas9 based gene editing in prostate cancer.

Gene therapy is highly suited for prostate cancer (PC). Metal-organic-frameworks (MOFs) are potential gene delivery systems. Target-specific cytoplasmic and nuclear knockdown in host gene expression using ZIF-C is shown for the first time through RNAi and CRISPR/Cas9 based gene editing in PC cells. A green tea phytochemical coating enhances intracellular delivery.

Machine-learning approach expands the repertoire of anti-CRISPR protein families.

The CRISPR-Cas are adaptive bacterial and archaeal immunity systems that have been harnessed for the development of powerful genome editing and engineering tools. In the incessant host-parasite arms race, viruses evolved multiple anti-defense mechanisms including diverse anti-CRISPR proteins (Acrs) that specifically inhibit CRISPR-Cas and therefore have enormous potential for application as modulators of genome editing tools. Most Acrs are small and highly variable proteins which makes their bioinformatic prediction a formidable task. We present a machine-learning approach for comprehensive Acr prediction. The model shows high predictive power when tested against an unseen test set and was employed to predict 2,500 candidate Acr families. Experimental validation of top candidates revealed two unknown Acrs (AcrIC9, IC10) and three other top candidates were coincidentally identified and found to possess anti-CRISPR activity. These results substantially expand the repertoire of predicted Acrs and provide a resource for experimental Acr discovery.

Critical Anti-CRISPR Locus Repression by a Bi-functional Cas9 Inhibitor.

Bacteriophages must rapidly deploy anti-CRISPR proteins (Acrs) to inactivate the RNA-guided nucleases that enforce CRISPR-Cas adaptive immunity in their bacterial hosts. Listeria monocytogenes temperate phages encode up to three anti-Cas9 proteins, with acrIIA1 always present. AcrIIA1 binds and inhibits Cas9 with its C-terminal domain; however, the function of its highly conserved N-terminal domain (NTD) is unknown. Here, we report that the AcrIIA1NTD is a critical transcriptional repressor of the strong anti-CRISPR promoter. A rapid burst of anti-CRISPR transcription occurs during phage infection and the subsequent negative feedback by AcrIIA1NTD is required for optimal phage replication, even in the absence of CRISPR-Cas immunity. In the presence of CRISPR-Cas immunity, full-length AcrIIA1 uses its two-domain architecture to act as a "Cas9 sensor," tuning acr expression according to Cas9 levels. Finally, we identify AcrIIA1NTD homologs in other Firmicutes and demonstrate that they have been co-opted by hosts as "anti-anti-CRISPRs," repressing phage anti-CRISPR deployment.

Machine learning predicts new anti-CRISPR proteins.

The increasing use of CRISPR-Cas9 in medicine, agriculture, and synthetic biology has accelerated the drive to discover new CRISPR-Cas inhibitors as potential mechanisms of control for gene editing applications. Many anti-CRISPRs have been found that inhibit the CRISPR-Cas adaptive immune system. However, comparing all currently known anti-CRISPRs does not reveal a shared set of properties for facile bioinformatic identification of new anti-CRISPR families. Here, we describe AcRanker, a machine learning based method to aid direct identification of new potential anti-CRISPRs using only protein sequence information. Using a training set of known anti-CRISPRs, we built a model based on XGBoost ranking. We then applied AcRanker to predict candidate anti-CRISPRs from predicted prophage regions within self-targeting bacterial genomes and discovered two previously unknown anti-CRISPRs: AcrllA20 (ML1) and AcrIIA21 (ML8). We show that AcrIIA20 strongly inhibits Streptococcus iniae Cas9 (SinCas9) and weakly inhibits Streptococcus pyogenes Cas9 (SpyCas9). We also show that AcrIIA21 inhibits SpyCas9, Streptococcus aureus Cas9 (SauCas9) and SinCas9 with low potency. The addition of AcRanker to the anti-CRISPR discovery toolkit allows researchers to directly rank potential anti-CRISPR candidate genes for increased speed in testing and validation of new anti-CRISPRs. A web server implementation for AcRanker is available online at http://acranker.pythonanywhere.com/.

Broad-spectrum anti-CRISPR proteins facilitate horizontal gene transfer.

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.

Potent CRISPR-Cas9 inhibitors from Staphylococcus genomes.

Anti-CRISPRs (Acrs) are small proteins that inhibit the RNA-guided DNA targeting activity of CRISPR-Cas enzymes. Encoded by bacteriophage and phage-derived bacterial genes, Acrs prevent CRISPR-mediated inhibition of phage infection and can also block CRISPR-Cas-mediated genome editing in eukaryotic cells. To identify Acrs capable of inhibiting Staphylococcus aureus Cas9 (SauCas9), an alternative to the most commonly used genome editing protein Streptococcus pyogenes Cas9 (SpyCas9), we used both self-targeting CRISPR screening and guilt-by-association genomic search strategies. Here we describe three potent inhibitors of SauCas9 that we name AcrIIA13, AcrIIA14, and AcrIIA15. These inhibitors share a conserved N-terminal sequence that is dispensable for DNA cleavage inhibition and have divergent C termini that are required in each case for inhibition of SauCas9-catalyzed DNA cleavage. In human cells, we observe robust inhibition of SauCas9-induced genome editing by AcrIIA13 and moderate inhibition by AcrIIA14 and AcrIIA15. We also find that the conserved N-terminal domain of AcrIIA13-AcrIIA15 binds to an inverted repeat sequence in the promoter of these Acr genes, consistent with its predicted helix-turn-helix DNA binding structure. These data demonstrate an effective strategy for Acr discovery and establish AcrIIA13-AcrIIA15 as unique bifunctional inhibitors of SauCas9.

Allosteric inhibition of CRISPR-Cas9 by bacteriophage-derived peptides.

BACKGROUND: CRISPR-Cas9 has been developed as a therapeutic agent for various infectious and genetic diseases. In many clinically relevant applications, constitutively active CRISPR-Cas9 is delivered into human cells without a temporal control system. Excessive and prolonged expression of CRISPR-Cas9 can lead to elevated off-target cleavage. The need for modulating CRISPR-Cas9 activity over time and dose has created the demand of developing CRISPR-Cas off switches. Protein and small molecule-based CRISPR-Cas inhibitors have been reported in previous studies. RESULTS: We report the discovery of Cas9-inhibiting peptides from inoviridae bacteriophages. These peptides, derived from the periplasmic domain of phage major coat protein G8P (G8PPD), can inhibit the in vitro activity of Streptococcus pyogenes Cas9 (SpCas9) proteins in an allosteric manner. Importantly, the inhibitory activity of G8PPD on SpCas9 is dependent on the order of guide RNA addition. Ectopic expression of full-length G8P (G8PFL) or G8PPD in human cells can inactivate the genome-editing activity of SpyCas9 with minimum alterations of the mutation patterns. Furthermore, unlike the anti-CRISPR protein AcrII4A that completely abolishes the cellular activity of CRISPR-Cas9, G8P co-transfection can reduce the off-target activity of co-transfected SpCas9 while retaining its on-target activity. CONCLUSION: G8Ps discovered in the current study represent the first anti-CRISPR peptides that can allosterically inactivate CRISPR-Cas9. This finding may provide insights into developing next-generation CRISPR-Cas inhibitors for precision genome engineering.

An enhanced assay to characterize anti-CRISPR proteins using a cell-free transcription-translation system.

The characterization of CRISPR-Cas immune systems in bacteria was quickly followed by the discovery of anti-CRISPR proteins (Acrs) in bacteriophages. These proteins block different steps of CRISPR-based immunity and, as some inhibit Cas nucleases, can offer tight control over CRISPR technologies. While Acrs have been identified against a few CRISPR-Cas systems, likely many more await discovery and application. Here, we report a rapid and scalable method for characterizing putative Acrs against Cas nucleases using an E. coli-derived cell-free transcription-translation system. Using known Acrs against type II Cas9 nucleases as models, we demonstrate how the method can be used to measure the inhibitory activity of individual Acrs in under two days. We also show how the method can overcome non-specific inhibition of gene expression observed for some Acrs. In total, the method should accelerate the interrogation and application of Acrs as CRISPR-Cas inhibitors.

Biochemical characterization of RNA-guided ribonuclease activities for CRISPR-Cas9 systems.

The majority of bacteria and archaea rely on CRISPR-Cas systems for RNA-guided, adaptive immunity against mobile genetic elements. The Cas9 family of type II CRISPR-associated DNA endonucleases generates programmable double strand breaks in the CRISPR-complementary DNA targets flanked by the PAM motif. Nowadays, CRISPR-Cas9 provides a set of powerful tools for precise genome manipulation in eukaryotes and prokaryotes. Recently, a few Cas9 orthologs have been reported to possess intrinsic CRISPR-guided, sequence-specific ribonuclease activities. These discoveries fundamentally expanded the targeting capability of CRISPR-Cas9 systems, and promise to provide new CRISPR tools to manipulate specific cellular RNA transcripts. Here we present a detailed method for the biochemical characterization of Cas9's RNA-targeting potential.

Anti-CRISPR AcrIIA5 Potently Inhibits All Cas9 Homologs Used for Genome Editing.

CRISPR-Cas9 systems provide powerful tools for genome editing. However, optimal employment of this technology will require control of Cas9 activity so that the timing, tissue specificity, and accuracy of editing may be precisely modulated. Anti-CRISPR proteins, which are small, naturally occurring inhibitors of CRISPR-Cas systems, are well suited for this purpose. A number of anti-CRISPR proteins have been shown to potently inhibit subgroups of CRISPR-Cas9 systems, but their maximal inhibitory activity is generally restricted to specific Cas9 homologs. Since Cas9 homologs vary in important properties, differing Cas9s may be optimal for particular genome-editing applications. To facilitate the practical exploitation of multiple Cas9 homologs, here we identify one anti-CRISPR, called AcrIIA5, that potently inhibits nine diverse type II-A and type II-C Cas9 homologs, including those currently used for genome editing. We show that the activity of AcrIIA5 results in partial in vivo cleavage of a single-guide RNA (sgRNA), suggesting that its mechanism involves RNA interaction.

Functional metagenomics-guided discovery of potent Cas9 inhibitors in the human microbiome.

CRISPR-Cas systems protect bacteria and archaea from phages and other mobile genetic elements, which use small anti-CRISPR (Acr) proteins to overcome CRISPR-Cas immunity. Because Acrs are challenging to identify, their natural diversity and impact on microbial ecosystems are underappreciated. To overcome this discovery bottleneck, we developed a high-throughput functional selection to isolate ten DNA fragments from human oral and fecal metagenomes that inhibit Streptococcus pyogenes Cas9 (SpyCas9) in Escherichia coli. The most potent Acr from this set, AcrIIA11, was recovered from a Lachnospiraceae phage. We found that AcrIIA11 inhibits SpyCas9 in bacteria and in human cells. AcrIIA11 homologs are distributed across diverse bacteria; many distantly-related homologs inhibit both SpyCas9 and a divergent Cas9 from Treponema denticola. We find that AcrIIA11 antagonizes SpyCas9 using a different mechanism than other previously characterized Type II-A Acrs. Our study highlights the power of functional selection to uncover widespread Cas9 inhibitors within diverse microbiomes.

Versatile transcription control based on reversible dCas9 binding.

The ability to control transcription in a time-dependent manner in vitro promises numerous applications in molecular biology and nanotechnology. Here we demonstrate an approach that enables precise, independent control over the production of multiple RNA transcripts in vitro using single guide RNA (sgRNA)-directed transcription blockades by catalytically dead Streptococcus pyogenes CRISPR-Cas9 enzyme (dCas9). We show that when bound to a DNA template, the dCas9:sgRNA complex forms a robust blockade to transcription by RNA polymerases (RNAPs) from bacteriophages SP6, T3, and T7 (>99.5% efficiency), and a partial blockade to transcription by Escherichia coli RNAP (∼70% efficiency). We find that all three bacteriophage RNAPs dissociate from the DNA template upon encountering the dCas9 blockade, while E. coli RNAP stays bound for at least the 90-min duration of our experiments. The blockade maintains >95% efficiency when four mismatches are introduced into the 5' end of the sgRNA target sequence. Notably, when using such a mismatched blockade, production of specific RNA species can be activated on demand by addition of a double-stranded competitor DNA perfectly matching the sgRNA. This strategy enables the independent production of multiple RNA species in a temporally controlled fashion from the same DNA template, demonstrating a new approach for transcription control.

Inhibition of CRISPR-Cas9 ribonucleoprotein complex assembly by anti-CRISPR AcrIIC2.

CRISPR-Cas adaptive immune systems function to protect bacteria from invasion by foreign genetic elements. The CRISPR-Cas9 system has been widely adopted as a powerful genome-editing tool, and phage-encoded inhibitors, known as anti-CRISPRs, offer a means of regulating its activity. Here, we report the crystal structures of anti-CRISPR protein AcrIIC2Nme alone and in complex with Nme1Cas9. We demonstrate that AcrIIC2Nme inhibits Cas9 through interactions with the positively charged bridge helix, thereby preventing sgRNA loading. In vivo phage plaque assays and in vitro DNA cleavage assays show that AcrIIC2Nme mediates its activity through a large electronegative surface. This work shows that anti-CRISPR activity can be mediated through the inhibition of Cas9 complex assembly.