Inhibition of CRISPR-Cas9 with Bacteriophage Proteins

There is a continuously expanding gap between predicted phage gene sequences and their corresponding functions, which has largely hampered the development of phage therapy. Previous studies reported several phage proteins that could interfere with the intracellular processes of the host to obtain efficient infection. But few phage proteins that protect host against phage infection have been identified and characterized in detail. Here, we isolate a phage, vB_Pae_QDWS, capable of infecting Pseudomonas aeruginosa PAO1 and report that its encoded Gp21 protein protects PAO1 against phage infection. Expression of Gp21 regulates bacterial quorum sensing with an inhibitory effect in low cell density and an activation effect in high cell density. By testing the type IV pilus (TFP)-mediated twitching motility and transmission electron microscopy analysis, Gp21 was found to decrease the pilus synthesis. Further, by constructing the TFP synthesis gene pilB mutant and performing adsorption and phage resistance assay, we demonstrated that the Gp21 protein could block phage infection via decreasing the TFP-mediated phage adsorption. Gp21 is a novel protein that inhibits phage efficacy against bacteria. The study deepens our understanding of phage-host interactions. IMPORTANCE The majority of the annotated phage genes are currently deposited as "hypothetical protein" with unknown function. Research has revealed that some phage proteins serve to inhibit or redirect the host intracellular processes for phage infection. Conversely, we report a phage encoded protein Gp21 that protects the host against phage infection. The pathways that Gp21 involved in antiphage defense in Pseudomonas aeruginosa PAO1 interfere with quorum sensing and decrease type IV pilus-mediated phage adsorption. Gp21 is a novel protein with a low sequence homology with other reported twitching inhibitory proteins. As a lytic phage-derived protein, Gp21 expression protects P. aeruginosa PAO1 from reinfection by phage vB_Pae_QDWS, which may explain the well-known pseudolysogeny caused by virulent phages. Our discoveries provide valuable new insight into phage-host evolutionary dynamics.

Viral Anti-CRISPR Tactics: No Success without Sacrifice

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

Studies on phage development: I. An acridine-sensitive clock

Phages have exerted severe evolutionary pressure on prokaryotes over billions of years, resulting in major rearrangements. Without every enzyme involved in the phage-bacterium interaction being examined; bacteriophages cannot be used in practical applications. Numerous studies conducted in the past few years have uncovered a huge variety of bacterial antiphage defense systems; nevertheless, the mechanisms of most of these systems are not fully understood. Understanding the interactions between bacteriophage and bacterial proteins is important for efficient host cell infection. Phage proteins involved in these bacteriophage-host interactions often arise immediately after infection. Here, we review the main groups of phage enzymes involved in the first stage of viral infection and responsible for the degradation of the bacterial membrane. These include polysaccharide depolymerases (endosialidases, endorhamnosidases, alginate lyases, and hyaluronate lyases), and peptidoglycan hydrolases (ectolysins and endolysins). Host target proteins are inhibited, activated, or functionally redirected by the phage protein. These interactions determine the phage infection of bacteria. Proteins of interest are holins, endolysins, and spanins, which are responsible for the release of progeny during the phage lytic cycle. This review describes the main bacterial and phage enzymes involved in phage infection and analyzes the therapeutic potential of bacteriophage-derived proteins.

Was it always your plan to become a microbiologist? I imagine in school you heard about not only the heroes of microbiology but also the great French scientists in bacterial genetics and gene expression. This seems almost like a predestined path? Charpentier: Yes, I am not sure whether this was my destiny at first. I was always interested early on, starting at six years old, in going to university because of the age difference with my oldest sister. When I was in the second year of primary school, she was going to university. Since that time, I wanted to go to university, too.

Caspase recruitment domains (CARDs) and pyrin domains are important facilitators of inflammasome activity and pyroptosis. Upon pathogen recognition by NLR proteins, CARDs recruit and activate caspases, which, in turn, activate gasdermin pore forming proteins to and induce pyroptotic cell death. Here we show that CARD-like domains are present in defense systems that protect bacteria against phage. The bacterial CARD is essential for protease-mediated activation of certain bacterial gasdermins, which promote cell death once phage infection is recognized. We further show that multiple anti-phage defense systems utilize CARD-like domains to activate a variety of cell death effectors. We find that these systems are triggered by a conserved immune evasion protein that phages use to overcome the bacterial defense system RexAB, demonstrating that phage proteins inhibiting one defense system can activate another. We also detect a phage protein with a predicted CARD-like structure that can inhibit the CARD-containing bacterial gasdermin system. Our results suggest that CARD domains represent an ancient component of innate immune systems conserved from bacteria to humans, and that CARD-dependent activation of gasdermins is conserved in organisms across the tree of life.

CRISPR-Cas9 systems have revolutionized biotechnology, creating diverse new opportunities for biomedical research and therapeutic genome and epigenome editing. Despite the abundance of bacterial CRISPR-Cas9 systems, relatively few are effective in human cells, limiting the overall potential of CRISPR technology. To expand the CRISPR-Cas toolbox, we characterized a set of type II CRISPR-Cas9 systems from select bacterial genera and species encoding diverse Cas9s. Four systems demonstrated robust and specific gene repression in human cells when used as nuclease-null dCas9s fused with a KRAB domain and were also highly active nucleases in human cells. These systems have distinct protospacer adjacent motifs (PAMs), including AT-rich motifs and sgRNA features orthogonal to the commonly used Staphylococcus aureus and Streptococcus pyogenes Cas9s. Additionally, we assessed gene activation when fused with the p300 catalytic domain. Notably, S. uberis Cas9 performed competitively against benchmarks with promising repression, activation, nuclease, and base editing activity. This study expands the CRISPR-Cas9 repertoire, enabling effective genome and epigenome editing for diverse applications.

PB1-like phages belong to Pbunavirus and are widespread in various environments. This group of phages is a promising candidate for treating human or animal infectious diseases caused by antibiotic-resistant P. aeruginosa. The lipopolysaccharide (LPS) has been identified as the receptor of different PB1-like phages, while little is known about the receptor-binding proteins (RBPs) of these phages. We constructed the tail fiber protein (gp50) of a PB1-like phage, PHW2, and its C- or N-terminus truncation variants to identify its role during the phage infection. The anti-gp50(453-964) antibody showed a similar effect to the antibody against gp50 in blocking the phage infection. The protein competition and cell binding assays showed that the gp50(1-451) doesn't exhibit an effect on the adsorption of the host cells. These results indicated that the C-terminus of gp50 is the essential region that mediates phage PHW2 adsorption and infection.

The impact of phage infection on the host cell is severe. In order to take over the cellular machinery, some phage proteins were produced to shut off the host biosynthesis early in the phage infection. The discovery and identification of these phage-derived inhibitors have a significant prospect of application in antibacterial treatment. This work presented a phage protein, gp70.1, with non-specific inhibitory effects on Pseudomonas aeruginosa and Escherichia coli. Gp70.1 was encoded by early gene – orf 70.1 from P. aeruginosa phage PaP3. The P. aeruginosa with a plasmid encoding gp70.1 showed with delayed growth and had the appearance of a small colony. The combination of multifaceted analysis including microarray-based transcriptomic analysis, RT-qPCR, nuclear magnetic resonance (NMR) spectroscopy-based metabolomics and phenotype experiments were performed to investigate the effects of gp70.1 on P. aeruginosa. A total of 178 genes of P. aeruginosa mainly involved in extracellular function and metabolism were differentially expressed in the presence of gp70.1 at three examined time points. Furthermore, our results indicated that gp70.1 had an extensive impact on the extracellular phenotype of P. aeruginosa, such as motility, pyocyanin, extracellular protease, polysaccharide, and cellulase. For the metabolism of P. aeruginosa, the main effect of gp70.1 was the reduction of amino acid consumption. Finally, the RNA polymerase sigma factor RpoS was identified as a potential cellular target of gp70.1. Gp70.1 was the first bacterial inhibitor identified from Pseudomonas aeruginosa phage PaP3. It was also the first phage protein that interacted with the global regulator RpoS of bacteria. Our results indicated the potential value of gp70.1 in antibacterial applications. This study preliminarily revealed the biological function of gp70.1 and provided a reference for the study of other phage genes sharing similarities with orf70.1.

High content analysis platform for optimization of lipid mediated CRISPR-Cas9 delivery strategies in human cells

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein-9 nuclease (Cas9) system is a new genome editing tool developed from the bacterial (Streptococcus pyogenes) adaptive immune mechanism. The CRISPR-Cas9 system sequence-specifically cleaves target sequence under the guide from single guide RNA (sgRNA) and the presence of protospacer-adjacent motif (PAM) in the target sequence. Another genome editing tool was recently developed and was named as the NgAgo-gDNA system. The NgAgo nuclease cleaves target sequence by binding to 5’-phophorylated 24-nucleotide single-strand guide DNA (gDNA). Here we have briefly introduced both the CRISPR-Cas9 and NgAgo-gDNA systems and compared them in their genome editing conditions, cleavage fidelity and efficiency as well as their application area. The NgAgo-gDNA system may have advantages in editing animal genomes due to the availability and convenience of artificially synthesized gDNAs. The NgAgo-gDNA system also benefits multiple target genes and shows higher efficiency in editing GC-rich genes with higher editing fidelity and less target sequence restriction. However, the transfected gDNA may also be integrated into an organism genome, which may lead to unexpected mutations or ethic issues. On the other hand, the current study showed that the best cleavage activity of NgAgo was observed at 37oC, which may restrict its application in some of organisms. In addition, the NgAgo-gDNA system may not be easily applied to edit plant genomes due to the unavailability of gDNA in plants. Thus, the CRISPR-Cas9 system could not be replaced by the NgAgo-gDNA system and both systems may have their own application potential in gene functional annotation and organism improvement as well as disease control. The NgAgo-gDNA system may have its advantages in human/animal genome editing whereas the CRISPR-Cas9 system has been proven to benefit plant genome modification.

RNA-guided nucleases such as CRISPR-Cas9 systems have revolutionized genome engineering by enabling programmable DNA modifications. Although structure-guided and evolution-derived high-fidelity Cas9 variants improve target specificity, they often compromise on-target activity or constrain guide RNA (gRNA) design. We performed head-to-head comparisons of OpenCRISPR-1 and Cas9 in human cells using amplicon sequencing, multiplex Digenome-seq, and off-target validation by targeted sequencing. Editing activity was assessed across 28 endogenous loci in HEK293T cells and further evaluated in human induced pluripotent stem cells (iPSCs) and MRC-5 fibroblasts. To test clinically relevant delivery, Cas9 and OpenCRISPR-1 ribonucleoproteins were delivered using engineered virus-like particles (eVLPs). We also generated OpenCRISPR-based prime editors, OpenCRISPR-PE2 and OpenCRISPR-PE7, and compared them with PE2max and PE7 using pegRNAs and engineered epegRNAs. Here, we show that OpenCRISPR-1, an AI-designed, Cas9-like nuclease, retains Cas9-level editing efficiency across multiple genomic loci while significantly reducing off-target mutations. Using multiplex Digenome-seq and targeted deep sequencing, OpenCRISPR-1 exhibits up to a 553-fold reduction in off-target mutations compared to Cas9 and achieves off-target indices that match or surpass those of high-fidelity Cas9 variants. OpenCRISPR-1 also sustains robust editing across diverse gRNA formats (GX19, gX19, and gX20), highlighting its enhanced versatility. Furthermore, converting OpenCRISPR-1 into a prime editor yields comparable editing efficiencies while lowering the relative specificity ratio by up to 97%. These findings establish generative AI-guided protein design as a powerful strategy to overcome the specificity-efficiency trade-off, expanding the genome editing toolkit for both research and therapeutic use, and ushering in a new era of rational protein design.

To correct a DNA mutation in the human genome for gene therapy, homology-directed repair (HDR) needs to be specific and have the lowest off-target effects to protect the human genome from deleterious mutations. Zinc finger nucleases, transcription activator-like effector nuclease (TALEN) and CRISPR-CAS9 systems have been engineered and used extensively to recognize and modify specific DNA sequences. Although TALEN and CRISPR/CAS9 could induce high levels of HDR in human cells, their genotoxicity was significantly higher. Here, we report the creation of a monomeric endonuclease that can recognize at least 33 bp by fusing the DNA-recognizing domain of TALEN (TALE) to a re-engineered homing endonuclease I-SceI. After sequentially re-engineering I-SceI to recognize 18 bp of the human β-globin sequence, the re-engineered I-SceI induced HDR in human cells. When the re-engineered I-SceI was fused to TALE (TALE-ISVB2), the chimeric endonuclease induced the same HDR rate at the human β-globin gene locus as that induced by TALEN, but significantly reduced genotoxicity. We further demonstrated that TALE-ISVB2 specifically targeted at the β-globin sequence in human hematopoietic stem cells. Therefore, this monomeric endonuclease has the potential to be used in therapeutic gene targeting in human cells.

Although numerous phage defense systems have recently been discovered in bacteria, how these systems defend against phage propagation or sense phage infections remains unclear. The Escherichia coli AbpAB defense system targets several lytic and lysogenic phages harboring DNA genomes. A phage-encoded single-stranded DNA-binding protein, Gp32, activates this system similar to other phage defense systems such as Retron-Eco8, Hachiman, ShosTA, Nhi, and Hna. DNA replication inhibitors or defects in DNA repair factors activate the AbpAB system, even without phage infection. This is one of the few examples of activating phage defense systems without phage infection or proteins. The AbpAB defense system may be activated by sensing specific DNA-protein complexes.