anti-CRISPR Proteins Research Articles

Identification and Optimization of Novel Small-Molecule Cas9 Inhibitors by Cell-Based High-Throughput Screening

CRISPR/Cas9 has revolutionized several areas of life science; however, methods to control the Cas9 activity are needed for both scientific and therapeutic applications. Anti-CRISPR proteins are known to inhibit the CRISPR/Cas adaptive immunity; however, in vivo delivery of such proteins is problematic. Instead, small-molecule Cas9 inhibitors could serve as useful tools due to their permeable, proteolytically stable, and non-immunogenic nature. Here, we identified a small-molecule ligand with anti-CRISPR/Cas9 activity through a high-throughput screening utilizing an Escherichia coli selection system. Extensive structure-activity relationship studies, which involved a deconstruction-reconstruction strategy, resulted in a range of analogues with significant improvements in the inhibitory activity. Based on NMR and electrophoretic mobility shift assays, we propose that the inhibitory action of these compounds likely results from direct binding to apo-Cas9, preventing Cas9:gRNA complex formation. These molecules may find use as Cas9 modulators in various applications.

Strategies for High-Efficiency Mutation Using the CRISPR/Cas System.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated systems have revolutionized traditional gene-editing tools and are a significant tool for ameliorating gene defects. Characterized by high target specificity, extraordinary efficiency, and cost-effectiveness, CRISPR/Cas systems have displayed tremendous potential for genetic manipulation in almost any organism and cell type. Despite their numerous advantages, however, CRISPR/Cas systems have some inherent limitations, such as off-target effects, unsatisfactory efficiency of delivery, and unwanted adverse effects, thereby resulting in a desire to explore approaches to address these issues. Strategies for improving the efficiency of CRISPR/Cas-induced mutations, such as reducing off-target effects, improving the design and modification of sgRNA, optimizing the editing time and the temperature, choice of delivery system, and enrichment of sgRNA, are comprehensively described in this review. Additionally, several newly emerging approaches, including the use of Cas variants, anti-CRISPR proteins, and mutant enrichment, are discussed in detail. Furthermore, the authors provide a deep analysis of the current challenges in the utilization of CRISPR/Cas systems and the future applications of CRISPR/Cas systems in various scenarios. This review not only serves as a reference for improving the maturity of CRISPR/Cas systems but also supplies practical guidance for expanding the applicability of this technology.

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.

Interaction of Bare dSpCas9, Scaffold gRNA, and Type II Anti-CRISPR Proteins Highly Favors the Control of Gene Expression in the Yeast S. cerevisiae.

Type II CRISPR-(d)SpCas9 and anti-CRISPR proteins (AcrIIs) show evidence of coevolution and competition for survival between bacteria and phages. In biotechnology, CRISPR-(d)SpCas9 is utilized for gene editing and transcriptional regulation. Moreover, its activity is controlled by AcrIIs. However, studies of dSpCas9/AcrII-based transcription regulation in Saccharomyces cerevisiae are rare. In this work, we used dSpCas9 as a template to engineer new transcription activators. We found that the most performant activation system requires the use of bare dSpCas9 in conjunction with scaffold gRNA (scRNA). This means that activation domains shall not be fused to dSpCas9 but rather interact with scRNA. We showed that a low amount of sgRNA is not a limiting factor in dSpCas9-driven transcription regulation. Moreover, a high quantity of sgRNA does not improve, generally, activation (and repression) efficiency. Importantly, we analyzed the performance of AcrIIA2, AcrIIA4, and AcrIIA5 in S. cerevisiae in depth. AcrIIA4 is the strongest of the three AcrIIs and also the only one able to induce high inhibition at low concentrations. However, the activation domains fused to dSpCas9 hindered interactions with the AcrIIs as well and limited their control of gene transcription regulation, confirming that bare dSpCas9 is the best solution for building synthetic genetic networks in yeast.

Molecular basis of transcriptional repression of anti-CRISPR by anti-CRISPR-associated 2.

CRISPR-Cas systems are well known host defense mechanisms that are conserved in bacteria and archaea. To counteract CRISPR-Cas systems, phages and viruses have evolved to possess multiple anti-CRISPR (Acr) proteins that can inhibit the host CRISPR-Cas system via different strategies. The expression of acr genes is controlled by anti-CRISPR-associated (Aca) proteins that bind toan upstream promoter and regulate the expression of acr genes during transcription. Although the role of Aca as a transcriptional repressor has been demonstrated, the mechanism of action of Aca has not been determined. Here, the molecular mechanism underlying the Aca2-mediated transcriptional control of acr genes was elucidated by determining the crystal structure of Aca2 from Oceanimonas smirnovii at a high resolution of 1.92 Å. Aca2 forms a dimer in solution, and dimerization of Aca2 is critical for specific promoter binding. The promoter-binding strategy of dimeric Aca2 was also revealed by performing mutagenesis studies. The atomic structure of the Aca family shown in this study provides insights into the fine regulation of host defense and immune-escape mechanisms and also demonstrates the conserved working mechanism of the Aca family.

Ecology and Evolution of Phages Encoding Anti-Crispr Proteins

Ecology and Evolution of Phages Encoding Anti-Crispr Proteins

In Silico Approaches for Prediction of Anti-CRISPR Proteins

In Silico Approaches for Prediction of Anti-CRISPR Proteins

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.

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.

Orthogonal CRISPR-Cas tools for genome editing, inhibition, and CRISPR recording in zebrafish embryos.

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas universe continues to expand. The type II CRISPR-Cas system from Streptococcus pyogenes (SpyCas9) is the most widely used for genome editing due to its high efficiency in cells and organisms. However, concentrating on a single CRISPR-Cas system imposes limits on target selection and multiplexed genome engineering. We hypothesized that CRISPR-Cas systems originating from different bacterial species could operate simultaneously and independently due to their distinct single-guide RNAs (sgRNAs) or CRISPR-RNAs (crRNAs), and protospacer adjacent motifs (PAMs). Additionally, we hypothesized that CRISPR-Cas activity in zebrafish could be regulated through the expression of inhibitory anti-CRISPR (Acr) proteins. Here, we use a simple mutagenesis approach to demonstrate that CRISPR-Cas systems from S. pyogenes (SpyCas9), Streptococcus aureus (SauCas9), Lachnospiraceae bacterium (LbaCas12a, previously known as LbCpf1) are orthogonal systems capable of operating simultaneously in zebrafish. CRISPR systems from Acidaminococcus sp. (AspCas12a, previously known as AsCpf1) and Neisseria meningitidis (Nme2Cas9) were also active in embryos. We implemented multichannel CRISPR recording using three CRISPR systems and show that LbaCas12a may provide superior information density compared with previous methods. We also demonstrate that type II Acrs (anti-CRISPRs) are effective inhibitors of SpyCas9 in zebrafish. Our results indicate that at least five CRISPR-Cas systems and two anti-CRISPR proteins are functional in zebrafish embryos. These orthogonal CRISPR-Cas systems and Acr proteins will enable combinatorial and intersectional strategies for spatiotemporal control of genome editing and genetic recording in animals.

Self-inactivating, all-in-one AAV vectors for precision Cas9 genome editing via homology-directed repair in vivo

Adeno-associated virus (AAV) vectors are important delivery platforms for therapeutic genome editing but are severely constrained by cargo limits. Simultaneous delivery of multiple vectors can limit dose and efficacy and increase safety risks. Here, we describe single-vector, ~4.8-kb AAV platforms that express Nme2Cas9 and either two sgRNAs for segmental deletions, or a single sgRNA with a homology-directed repair (HDR) template. We also use anti-CRISPR proteins to enable production of vectors that self-inactivate via Nme2Cas9 cleavage. We further introduce a nanopore-based sequencing platform that is designed to profile rAAV genomes and serves as a quality control measure for vector homogeneity. We demonstrate that these platforms can effectively treat two disease models [type I hereditary tyrosinemia (HT-I) and mucopolysaccharidosis type I (MPS-I)] in mice by HDR-based correction of the disease allele. These results will enable the engineering of single-vector AAVs that can achieve diverse therapeutic genome editing outcomes.

Structural basis for anti-CRISPR repression mediated by bacterial operon proteins Aca1 and Aca2

It has been shown that phages have evolved anti-CRISPR (Acr) proteins to inhibit host CRISPR-Cas systems. Most acr genes are located upstream of anti-CRISPR-associated (aca) genes, which is instrumental for identifying these acr genes. Thus far, eight Aca families (Aca1–Aca8) have been identified, all proteins of which share low sequence homology and bind to different target DNA sequences. Recently, Aca1 and Aca2 proteins were discovered to function as repressors by binding to acr-aca promoters, thus implying a potential anti-anti-CRISPR mechanism. However, the structural basis for the repression roles of Aca proteins is still unknown. Here, we elucidated apo-structures of Aca1 and Aca2 proteins and their complex structures with their cognate operator DNA in two model systems, the Pseudomonas phage JBD30 and the Pectobacterium carotovorum template phage ZF40. In combination with biochemical and cellular assays, our study unveils dimerization and DNA-recognition mechanisms of Aca1 and Aca2 family proteins, thus revealing the molecular basis for Aca1-and Aca2-mediated anti-CRISPR repression. Our results also shed light on understanding the repression roles of other Aca family proteins and autoregulation roles of acr-aca operons.

Rapidly Characterizing CRISPR-Cas13 Nucleases Using Cell-Free Transcription-Translation Systems.

Cell-free transcription-translation (TXTL) systems produce RNAs and proteins from added DNA. By coupling their production to a biochemical assay, these biomolecules can be rapidly and scalably characterized without the need for purification or cell culturing. Here, we describe how TXTL can be applied to characterize Cas13 nucleases from Type VI CRISPR-Cas systems. These nucleases employ guide RNAs to recognize complementary RNA targets, leading to the nonspecific collateral cleavage of nearby RNAs. In turn, RNA targeting by Cas13 has been exploited for numerous applications, including in vitro diagnostics, programmable gene silencing in eukaryotes, and sequence-specific antimicrobials. As part of the described method, we detail how to set up TXTL assays to measure on-target and collateral RNA cleavage by Cas13 as well as how to assay for putative anti-CRISPR proteins. Overall, the method should be useful for the characterization of Type VI CRISPR-Cas systems and their use in ranging applications.

Off-target effects of base editors: what we know and how we can reduce it.

The recently discovered CRISPR-Cas9 modification, base editors (BEs), is considered as one of the most promising tools for correcting disease-causing mutations in humans, since it allows point substitutions to be edited without generating double-stranded DNA breaks, and, therefore, with a significant decrease in non-specific activity. Until recently, this method was considered the safest, but at the same time, it is quite effective. However, recent studies of non-specific activity of BEs revealed that some of them lead to the formation of a huge number of off-targets in both DNA and RNA, occurring due to the nature of the Cas9-fused proteins used. In this review article, we have considered and combined data from numerous studies about the most commonly used and more described in detail APOBEC-based BEs and Target-AID version of CBE, as well as ABE7 and ABE8 with their basic modifications into TadA to improve BEs' specificity. In our opinion, modern advances in molecular genetics make it possible to dramatically reduce the off-target activity of base editors due to introducing mutations into the domains of deaminases or inhibition of Cas9 by anti-CRISPR proteins, which returns BEs to the leading position in genome editing technologies.

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.

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.

Crystal structure of the anti-CRISPR repressor Aca2

Bacteria use adaptive CRISPR-Cas immune mechanisms to protect from invasion by bacteriophages and other mobile genetic elements. In response, bacteriophages and mobile genetic elements have co-evolved anti-CRISPR proteins to inhibit the bacterial defense. We and others have previously shown that anti-CRISPR associated (Aca) proteins can regulate this anti-CRISPR counter-attack. Here, we report the first structure of an Aca protein, the Aca2 DNA-binding transcriptional autorepressor from Pectobacterium carotovorum bacteriophage ZF40, determined to 1.34 Å. Aca2 presents a conserved N-terminal helix-turn-helix DNA-binding domain and a previously uncharacterized C-terminal dimerization domain. Dimerization positions the Aca2 recognition helices for insertion into the major grooves of target DNA, supporting its role in regulating anti-CRISPRs. Furthermore, database comparisons identified uncharacterized Aca2 structural homologs in pathogenic bacteria, suggesting that Aca2 represents the first characterized member of a more widespread family of transcriptional regulators.

Anti-CRISPR proteins could help put the brakes on gene editing

Anti-CRISPR proteins could help put the brakes on gene editing

Structure-based functional mechanisms and biotechnology applications of anti-CRISPR proteins.

CRISPR loci and Cas proteins provide adaptive immunity in prokaryotes against invading bacteriophages and plasmids. In response, bacteriophages have evolved a broad spectrum of anti-CRISPR proteins (anti-CRISPRs) to counteract and overcome this immunity pathway. Numerous anti-CRISPRs have been identified to date, which suppress single-subunit Cas effectors (in CRISPR class 2, type II, V and VI systems) and multisubunit Cascade effectors (in CRISPR class 1, type I and III systems). Crystallography and cryo-electron microscopy structural studies of anti-CRISPRs bound to effector complexes, complemented by functional experiments in vitro and in vivo, have identified four major CRISPR-Cas suppression mechanisms: inhibition of CRISPR-Cas complex assembly, blocking of target binding, prevention of target cleavage, and degradation of cyclic oligonucleotide signalling molecules. In this Review, we discuss novel mechanistic insights into anti-CRISPR function that have emerged from X-ray crystallography and cryo-electron microscopy studies, and how these structures in combination with function studies provide valuable tools for the ever-growing CRISPR-Cas biotechnology toolbox, to be used for precise and robust genome editing and other applications.

Structural Investigation of Self-Assembly and Target Binding of Anti-CRISPR AcrIIC2.

Anti-CRISPR (Acr) proteins are phage-borne inhibitors of the CRISPR-Cas immune system in archaea and bacteria. AcrIIC2 from prophages of Neisseria meningitidis disables the nuclease activity of type II-C Cas9, such that dimeric AcrIIC2 associates with the bridge helix (BH) region of Cas9 to compete with guide RNA loading. AcrIIC2 in solution readily assembles into oligomers of variable lengths, but the oligomeric states are not clearly understood. In this study, we investigated the dynamic assembly of AcrIIC2 oligomers, and identified key interactions underlying the self-association. We report that AcrIIC2 dimers associate into heterogeneous high-order oligomers with the equilibrium dissociation constant K D ∼8 μM. Oligomerization is driven by electrostatic interactions between charged residues, and rational mutagenesis produces a stable AcrIIC2 dimer with intact Cas9 binding. Remarkably, the BH peptide of Cas9 is unstructured in solution, and undergoes a coil-to-helix transition upon AcrIIC2 binding, revealing a unique folding-upon-binding mechanism for Acr recognition.