AcrIIA7 hijacks tracrRNA to block CRISPR-Cas system.

Box C/D ribonucleoprotein (RNP) complexes direct the nucleotide-specific 2'-O-methylation of ribonucleotide sugars in target RNAs. In vitro assembly of an archaeal box C/D sRNP using recombinant core proteins L7, Nop56/58 and fibrillarin has yielded an RNA:protein enzyme that guides methylation from both the terminal box C/D core and internal C'/D' RNP complexes. Reconstitution of sRNP complexes containing only box C/D or C'/D' motifs has demonstrated that the terminal box C/D RNP is the minimal methylation-competent particle. However, efficient ribonucleotide 2'-O-methylation requires that both the box C/D and C'/D' RNPs function within the full-length sRNA molecule. In contrast to the eukaryotic snoRNP complex, where the core proteins are distributed asymmetrically on the box C/D and C'/D' motifs, all three archaeal core proteins bind both motifs symmetrically. This difference in core protein distribution is a result of altered RNA-binding capabilities of the archaeal and eukaryotic core protein homologs. Thus, evolution of the box C/D nucleotide modification complex has resulted in structurally distinct archaeal and eukaryotic RNP particles.

RNA-binding proteins (RBPs) form ribonucleoprotein (RNP) complexes that play crucial roles in RNA processing for gene regulation. The angiosperm sieve tube system contains a unique population of transcripts, some of which function as long-distance signaling agents involved in regulating organ development. These phloem-mobile mRNAs are translocated as RNP complexes. One such complex is based on a phloem RBP named Cucurbita maxima RNA-binding protein 50 (CmRBP50), a member of the polypyrimidine track binding protein family. The core of this RNP complex contains six additional phloem proteins. Here, requirements for assembly of this CmRBP50 RNP complex are reported. Phosphorylation sites on CmRBP50 were mapped, and then coimmunoprecipitation and protein overlay studies established that the phosphoserine residues, located at the C terminus of CmRBP50, are critical for RNP complex assembly. In vitro pull-down experiments revealed that three phloem proteins, C. maxima phloem protein 16, C. maxima GTP-binding protein, and C. maxima phosphoinositide-specific phospholipase-like protein, bind directly with CmRBP50. This interaction required CmRBP50 phosphorylation. Gel mobility-shift assays demonstrated that assembly of the CmRBP50-based protein complex results in a system having enhanced binding affinity for phloem-mobile mRNAs carrying polypyrimidine track binding motifs. This property would be essential for effective long-distance translocation of bound mRNA to the target tissues.

In measles virus (MV)-infected cells, the ribonucleoprotein (RNP) complex, comprised of the viral genome and the nucleocapsid (N) protein, phosphoprotein (P protein), and large protein, assembles at the perinuclear region and synthesizes viral RNAs. The cellular proteins involved in the formation of the RNP complex are largely unknown. In this report, we show that cofilin, an actin-modulating host protein, interacts with the MV N protein and aids in the formation of the RNP complex. Knockdown of cofilin using the short hairpin RNA reduces the formation of the RNP complex after MV infection and that of the RNP complex-like structure after plasmid-mediated expression of MV N and P proteins. A lower level of formation of the RNP complex results in the reduction of viral RNA synthesis. Cofilin phosphorylation on the serine residue at position 3, an enzymatically inactive form, is increased after MV infection and the phosphorylated form of cofilin is preferentially included in the complex. These results indicate that cofilin plays an important role in MV replication by increasing formation of the RNP complex and viral RNA synthesis. Many RNA viruses induce within infected cells the structure called the ribonucleoprotein (RNP) complex in which viral RNA synthesis occurs. It is comprised of the viral genome and proteins that include the viral RNA polymerase. The cellular proteins involved in the formation of the RNP complex are largely unknown. In this report, we show that cofilin, an actin-modulating host protein, binds to the measles virus (MV) nucleocapsid protein and plays an important role in the formation of the MV RNP complex and MV RNA synthesis. The level of the phosphorylated form of cofilin, enzymatically inactive, is increased after MV infection, and the phosphorylated form is preferentially associated with the RNP complex. Our findings determined with cofilin will help us better understand the mechanism by which the RNP complex is formed in virus-infected cells and develop new antiviral drugs targeting the RNP complex.

The Broad Institute Scores Another Victory in Its Battle with the University of California over the Patenting of CRIPSR

Widespread RNA-based cas regulation monitors crRNA abundance and anti-CRISPR proteins

BackgroundAnti-CRISPR proteins are potent modulators that inhibit the CRISPR-Cas immunity system and have huge potential in gene editing and gene therapy as a genome-editing tool. Extensive studies have shown that anti-CRISPR proteins are essential for modifying endogenous genes, promoting the RNA-guided binding and cleavage of DNA or RNA substrates. In recent years, identifying and characterizing anti-CRISPR proteins has become a hot and significant research topic in bioinformatics. However, as most anti-CRISPR proteins fall short in sharing similarities to those currently known, traditional screening methods are time-consuming and inefficient. Machine learning methods could fill this gap with powerful predictive capability and provide a new perspective for anti-CRISPR protein identification.ResultsHere, we present a novel machine learning ensemble predictor, called PreAcrs, to identify anti-CRISPR proteins from protein sequences directly. Three features and eight different machine learning algorithms were used to train PreAcrs. PreAcrs outperformed other existing methods and significantly improved the prediction accuracy for identifying anti-CRISPR proteins.ConclusionsIn summary, the PreAcrs predictor achieved a competitive performance for predicting new anti-CRISPR proteins in terms of accuracy and robustness. We anticipate PreAcrs will be a valuable tool for researchers to speed up the research process. The source code is available at: https://github.com/Lyn-666/anti_CRISPR.git.

CRISPR-Cas9 systems are bacterial adaptive immune systems that defend against infection by phages. Through the RNA-guided endonuclease activity of Cas9 they degrade double-stranded DNA with a protospacer adjacent motif (PAM) and sequences complementary to the guide RNA. Recently, two anti-CRISPR proteins (AcrIIA2 and AcrIIA4 from Listeria monocytogenes prophages) were identified, both of which inhibit Streptococcus pyogenes Cas9 (SpyCas9) and L. monocytogenes Cas9 activity in bacteria and human cells. However, the mechanism of AcrIIA2- or AcrIIA4-mediated Cas9 inhibition remains unknown. Here we report a crystal structure of SpyCas9 in complex with a single-guide RNA (sgRNA) and AcrIIA4. Our data show that AcrIIA2 and AcrIIA4 interact with SpyCas9 in a sgRNA-dependent manner. The structure reveals that AcrIIA4 inhibits SpyCas9 activity by structurally mimicking the PAM to occupy the PAM-interacting site in the PAM-interacting domain, thereby blocking recognition of double-stranded DNA substrates by SpyCas9. AcrIIA4 further inhibits the endonuclease activity of SpyCas9 by shielding its RuvC active site. Structural comparison reveals that formation of the AcrIIA4-binding site of SpyCas9 is induced by sgRNA binding. Our study reveals the mechanism of SpyCas9 inhibition by AcrIIA4, providing a structural basis for developing 'off-switch' tools for SpyCas9 to avoid unwanted genome edits within cells and tissues.

Bacteria and the viruses that infect them are engaged in an evolutionary arms race that has driven the development of bacterial defense mechanisms and phage countermeasures to those defense mechanisms. One instance of this evolutionary competition is the development of the CRISPR‐Cas system, which enables the recognition and destruction of phage DNA. In response to this defense, phages have evolved anti‐CRISPR proteins that inhibit CRISPR‐Cas activity and promote phage infection. It is thought that many anti‐CRISPR proteins exist in nature, but only a handful have been identified and their mechanism of inhibition characterized. The goal of this project is to understand the mechanism by which a recently identified anti‐CRISPR protein, AcrIIA11, inhibits Streptococcus pyogenes Cas9 (SpyCas9). To address this, we have performed in vitro DNA cleavage and protein binding assays with purified components. We have determined the following: AcrIIA11 effectively inhibits Cas9 cleavage at an AcrIIA11: Cas9 ratio of 8:1; AcrIIA11 interacts directly with the Cas9 protein; homologs of AcrIIA11 show a range of ability to inhibit Cas9 activity but do no inhibit as well as AcrIIA11; and the C‐terminus of AcrIIA11 is not required for binding to Cas9. In future studies, we will test AcrIIA11 interaction with a wider range of Cas9 orthologs. From these results, we have gained a broader understanding of how AcrIIA11 interacts with SpyCas9.Support or Funding InformationSeattle University Summer Undergraduate Fellowship (Peach Foundation)

CRISPR-Cas9 systems provide adaptive immunity in prokaryotes by targeting and cleaving invading phage DNA. In response, phages have evolved anti-CRISPR (Acr) proteins to inhibit Cas9 and evade this immune response. AcrIIA26 is a type II-A anti-CRISPR protein that inhibits Streptococcus pyogenes Cas9 (SpyCas9) DNA binding, but its molecular mechanism remains unclear. Here, we determined the 3.0 Å resolution cryo-EM structure of AcrIIA26 in complex with SpyCas9-single-guide RNA, revealing a dual inhibition mechanism. AcrIIA26 adopts a novel fold comprising a central β-sheet flanked by two α-helical bundles. The 5-helix bundle, which features a negatively charged surface whose shape mimics duplex DNA, occupies the same position as the protospacer adjacent motif (PAM) duplex in target-bound Cas9. This directly blocks PAM recognition by burying critical residues R1333 and R1335 in the PAM-interacting domain. Mutagenesis confirmed that residues E49 and D50 in AcrIIA26 are essential for this interaction. Simultaneously, the 4-helix bundle binds the Cas9 REC lobe and sterically prevents the conformational changes required for Cas9 activation, with mutation of AcrIIA26 F121 completely eliminating inhibitory activity. Structural comparisons reveal that despite diverse folds, multiple anti-CRISPRs convergently evolved to block PAM recognition, highlighting this as a critical vulnerability in Cas9 function. Our findings provide mechanistic insights into AcrIIA26 inhibition and offer a foundation for engineering improved Cas9 off-switches for genome editing applications.

Anti-CRISPR (Acr) proteins can evade CRISPR-Cas immunity, yet their molecular determinants remain poorly understood. This study aimed to uncover key features driving Acr activity, thereby advancing both fundamental knowledge and the rational design of robust CRISPR-based tools. We compiled a binary-encoded matrix of 761 InterPro-annotated domains and binding-site features for known Acr proteins. Seven feature ranking algorithms were applied to prioritize determinant features, and an incremental feature selection strategy, coupled with four distinct classifiers, was used to identify optimal subsets. Consensus key features were defined by intersecting the top subsets across all methods. Key identified features include the DUF2829 domain, the Lambda repressor-like domain and Sulfolobus islandicus virus proteins, the Cro/C1-type helix-turn-helix domain, phage protein, and replication initiator A. These findings illuminate novel structural modules and regulatory motifs that underpin Acr inhibition. This study provides critical theoretical support for deciphering Acr mechanisms and offers actionable insights for engineering next-generation CRISPR-Cas applications in clinical and biotechnological settings. The CRISPR system is a part of the antiviral immune defense initially discovered in bacteria and archaea. At present, the CRISPR system has become the cornerstone of genome editing technologies such as CRISPR-Cas9, widely used in clinical, agricultural, and biological research. Anti-CRISPR proteins are a group of proteins that inhibit the normal activity of CRISPR-Cas system in certain bacteria or archaea and avoid having the phages' genomes destroyed by the prokaryotic cells. The anti-CRISPR protein family has various components, but with similar functions to help exogenous DNA escape from the immune system. This study tried to uncover molecular mechanisms for anti-CRISPR proteins.

The Streptococcus pyogenes Cas9 protein (SpCas9), a component of CRISPR-based immune system in microbes, has become commonly utilized for genome editing. This nuclease forms a ribonucleoprotein (RNP) complex with guide RNA (gRNA) which induces Cas9 structural changes and triggers its cleavage activity. Here, electronic circular dichroism (ECD) spectroscopy was used to confirm the RNP formation and to determine its individual components. The ECD spectra had characteristic features differentiating Cas9 and gRNA, the former showed a negative/positive profile with maxima located at 221, 209 and 196 nm, while the latter revealed positive/negative/positive/negative pattern with bands observed at 266, 242, 222 and 209 nm, respectively. For the first time, the experimental ECD spectrum of the gRNA:Cas9 RNP complex is presented. It exhibits a bisignate positive/negative ECD couplet with maxima at 273 and 235 nm, and it differs significantly from individual spectrum of each RNP components. Additionally, the Cas9 protein and RNP complex retained biological activity after ECD measurements and they were able to bind and cleave DNA in vitro. Hence, we conclude that ECD spectroscopy can be considered as a quick and non-destructive method of monitoring conformational changes of the Cas9 protein as a result of Cas9 and gRNA interaction, and identification of the gRNA:Cas9 RNP complex.

Anti-CRISPR proteins (Acrs) block the activity of CRISPR-associated (Cas) proteins, either by inhibiting DNA interference or by preventing crRNA loading and complex formation. Although the main use of Acrs in genome engineering applications is to lower the cleavage activity of Cas proteins, they can also be instrumental for various other CRISPR-based applications. Here, we explore the genome editing potential of the thermoactive type II-C Cas9 variants from Geobacillus thermodenitrificans T12 (ThermoCas9) and Geobacillus stearothermophilus (GeoCas9) in Escherichia coli. We then demonstrate that the AcrIIC1 protein from Neisseria meningitidis robustly inhibits their DNA cleavage activity, but not their DNA binding capacity. Finally, we exploit these AcrIIC1:Cas9 complexes for gene silencing and base-editing, developing Acr base-editing tools. With these tools we pave the way for future engineering applications in mesophilic and thermophilic bacteria combining the activities of Acr and CRISPR-Cas proteins.

In their natural settings, CRISPR-Cas systems play crucial roles in bacterial and archaeal adaptive immunity to protect against phages and other mobile genetic elements, and they are also widely used as genome engineering technologies. Previously we discovered bacteriophage-encoded Cas9-specific anti-CRISPR (Acr) proteins that serve as countermeasures against host bacterial immunity by inactivating their CRISPR-Cas systems (A. Pawluk, N. Amrani, Y. Zhang, B. Garcia, et al., Cell 167:1829-1838.e9, 2016, https://doi.org/10.1016/j.cell.2016.11.017). We hypothesized that the evolutionary advantages conferred by anti-CRISPRs would drive the widespread occurrence of these proteins in nature (K. L. Maxwell, Mol Cell 68:8-14, 2017, https://doi.org/10.1016/j.molcel.2017.09.002; A. Pawluk, A. R. Davidson, and K. L. Maxwell, Nat Rev Microbiol 16:12-17, 2018, https://doi.org/10.1038/nrmicro.2017.120; E. J. Sontheimer and A. R. Davidson, Curr Opin Microbiol 37:120-127, 2017, https://doi.org/10.1016/j.mib.2017.06.003). We have identified new anti-CRISPRs using the same bioinformatic approach that successfully identified previous Acr proteins (A. Pawluk, N. Amrani, Y. Zhang, B. Garcia, et al., Cell 167:1829-1838.e9, 2016, https://doi.org/10.1016/j.cell.2016.11.017) against Neisseria meningitidis Cas9 (NmeCas9). In this work, we report two novel anti-CRISPR families in strains of Haemophilus parainfluenzae and Simonsiella muelleri, both of which harbor type II-C CRISPR-Cas systems (A. Mir, A. Edraki, J. Lee, and E. J. Sontheimer, ACS Chem Biol 13:357-365, 2018, https://doi.org/10.1021/acschembio.7b00855). We characterize the type II-C Cas9 orthologs from H. parainfluenzae and S. muelleri, show that the newly identified Acrs are able to inhibit these systems, and define important features of their inhibitory mechanisms. The S. muelleri Acr is the most potent NmeCas9 inhibitor identified to date. Although inhibition of NmeCas9 by anti-CRISPRs from H. parainfluenzae and S. muelleri reveals cross-species inhibitory activity, more distantly related type II-C Cas9s are not inhibited by these proteins. The specificities of anti-CRISPRs and divergent Cas9s appear to reflect coevolution of their strategies to combat or evade each other. Finally, we validate these new anti-CRISPR proteins as potent off-switches for Cas9 genome engineering applications.IMPORTANCE As one of their countermeasures against CRISPR-Cas immunity, bacteriophages have evolved natural inhibitors known as anti-CRISPR (Acr) proteins. Despite the existence of such examples for type II CRISPR-Cas systems, we currently know relatively little about the breadth of Cas9 inhibitors, and most of their direct Cas9 targets are uncharacterized. In this work we identify two new type II-C anti-CRISPRs and their cognate Cas9 orthologs, validate their functionality in vitro and in bacteria, define their inhibitory spectrum against a panel of Cas9 orthologs, demonstrate that they act before Cas9 DNA binding, and document their utility as off-switches for Cas9-based tools in mammalian applications. The discovery of diverse anti-CRISPRs, the mechanistic analysis of their cognate Cas9s, and the definition of Acr inhibitory mechanisms afford deeper insight into the interplay between Cas9 orthologs and their inhibitors and provide greater scope for exploiting Acrs for CRISPR-based genome engineering.

The programmable endonuclease activity and simple usage of CRISPR/Cas9 have revolutionized the field of genome editing. The binding of single guide RNA (sgRNA) by the Cas9 protein results in the formation of negatively charged ribonucleoprotein (RNP) complexes. The presence of this functional complex inside cells is imperative for the intended specific genome modifications. The direct intracellular delivery of Cas9/sgRNA RNP complexes is of great advantage. In this work, a compound library of sequence-defined oligo(ethylenamino) amides containing structural motifs for stable nanoparticle formation, cellular uptake, and endosomal release was used for the screening and development of suitable Cas9 RNP delivery vehicles. Lipid-containing oligoaminoamides (lipo-OAAs) were identified as the most efficient carriers for intracellular Cas9/sgRNA delivery and gene disruption. Fluorescence correlation spectroscopy measurements indicated that the lipo-OAAs only interact with sgRNA-loaded Cas9 protein, which suggests exclusive ionic interaction with the negatively charged RNPs. The type of contained fatty acid turned out to have a critical impact on the knock out efficiency: the presence of one hydroxy group in the fatty acid dramatically changes the properties and performance of the resulting Cas9/sgRNA lipo-OAA complexes. The lipo-OAA-containing hydroxy-stearic acid (OHSteA) was superior to the analogues with saturated or unsaturated fatty acids without hydroxylation; it formed smaller and more defined nanoparticles with Cas9/sgRNA and improved the cellular uptake and endosomal release, which altogether resulted in an increased nuclear association and the highest gene knock out levels. The efficient and adaptable delivery platform has high potential for the future development of therapeutics based on precise genome modifications.

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