(searched for: doi:10.1146/annurev-genet-022120-112523)
Frontiers in Microbiology, Volume 12; doi:10.3389/fmicb.2021.641317
Bacteriophages and their bacterial hosts are ancient organisms that have been co-evolving for billions of years. Some jumbo phages, those with a genome size larger than 200 kilobases, have recently been discovered to establish complex subcellular organization during replication. Here, we review our current understanding of jumbo phages that form a nucleus-like structure, or “Phage Nucleus,” during replication. The phage nucleus is made of a proteinaceous shell that surrounds replicating phage DNA and imparts a unique subcellular organization that is temporally and spatially controlled within bacterial host cells by a phage-encoded tubulin (PhuZ)-based spindle. This subcellular architecture serves as a replication factory for jumbo Pseudomonas phages and provides a selective advantage when these replicate in some host strains. Throughout the lytic cycle, the phage nucleus compartmentalizes proteins according to function and protects the phage genome from host defense mechanisms. Early during infection, the PhuZ spindle positions the newly formed phage nucleus at midcell and, later in the infection cycle, the spindle rotates the nucleus while delivering capsids and distributing them uniformly on the nuclear surface, where they dock for DNA packaging. During the co-infection of two different nucleus-forming jumbo phages in a bacterial cell, the phage nucleus establishes Subcellular Genetic Isolation that limits the potential for viral genetic exchange by physically separating co-infection genomes, and the PhuZ spindle causes Virogenesis Incompatibility, whereby interacting components from two diverging phages negatively affect phage reproduction. Thus, the phage nucleus and PhuZ spindle are defining cell biological structures that serve roles in both the life cycle of nucleus-forming jumbo phages and phage speciation.
Published: 3 June 2021
Proceedings of the National Academy of Sciences, Volume 118; doi:10.1073/pnas.2023202118
Despite remarkable strides in microbiome research, the viral component of the microbiome has generally presented a more challenging target than the bacteriome. This gap persists, even though many thousands of shotgun sequencing runs from human metagenomic samples exist in public databases, and all of them encompass large amounts of viral sequence data. The lack of a comprehensive database for human-associated viruses has historically stymied efforts to interrogate the impact of the virome on human health. This study probes thousands of datasets to uncover sequences from over 45,000 unique virus taxa, with historically high per-genome completeness. Large publicly available case-control studies are reanalyzed, and over 2,200 strong virus–disease associations are found.
ACS Synthetic Biology, Volume 10, pp 1245-1267; doi:10.1021/acssynbio.1c00107
Over the past decades, significant progress has been made in targeted cancer therapy. In precision oncology, molecular profiling of cancer patients enables the use of targeted cancer therapeutics. However, current diagnostic methods for molecular analysis of cancer are costly and require sophisticated equipment. Moreover, targeted cancer therapeutics such as monoclonal antibodies and small-molecule drugs may cause off-target effects and they are available for only a minority of cancer driver proteins. Therefore, there is still a need for versatile, efficient, and precise tools for cancer diagnostics and targeted cancer treatment. In recent years, the CRISPR-based genome and transcriptome engineering toolbox has expanded rapidly. Particularly, the RNA-targeting CRISPR-Cas13 system has unique biochemical properties, making Cas13 a promising tool for cancer diagnosis, therapy, and research. Cas13-based diagnostic methods allow early detection and monitoring of cancer markers from liquid biopsy samples without the need for complex instrumentation. In addition, Cas13 can be used for targeted cancer therapy through degrading and manipulating cancer-associated transcripts with high efficiency and specificity. Moreover, Cas13-mediated programmable RNA manipulation tools offer invaluable opportunities for cancer research, identification of drug-resistance mechanisms, and discovery of novel therapeutic targets. Here, we review and discuss the current use and potential applications of the CRISPR-Cas13 system in cancer diagnosis, therapy, and research. Thus, researchers will gain a deep understanding of CRISPR-Cas13 technologies, which have the potential to be used as next-generation cancer diagnostics and therapeutics.
Published: 25 May 2021
Proceedings of the National Academy of Sciences, Volume 118; doi:10.1073/pnas.2021291118
For Type I CRISPR-Cas systems, a mode of CRISPR adaptation named priming has been described. Priming allows specific and highly efficient acquisition of new spacers from DNA recognized (primed) by the Cascade-crRNA (CRISPR RNA) effector complex. Recognition of the priming protospacer by Cascade-crRNA serves as a signal for engaging the Cas3 nuclease–helicase required for both interference and primed adaptation, suggesting the existence of a primed adaptation complex (PAC) containing the Cas1–Cas2 adaptation integrase and Cas3. To detect this complex in vivo, we here performed chromatin immunoprecipitation with Cas3-specific and Cas1-specific antibodies using cells undergoing primed adaptation. We found that prespacers are bound by both Cas1 (presumably, as part of the Cas1–Cas2 integrase) and Cas3, implying direct physical association of the interference and adaptation machineries as part of PAC.
Genes, Volume 12; doi:10.3390/genes12060797
Genome-editing (GE) is having a tremendous influence around the globe in the life science community. Among its versatile uses, the desired modifications of genes, and more importantly the transgene (DNA)-free approach to develop genetically modified organism (GMO), are of special interest. The recent and rapid developments in genome-editing technology have given rise to hopes to achieve global food security in a sustainable manner. We here discuss recent developments in CRISPR-based genome-editing tools for crop improvement concerning adaptation, opportunities, and challenges. Some of the notable advances highlighted here include the development of transgene (DNA)-free genome plants, the availability of compatible nucleases, and the development of safe and effective CRISPR delivery vehicles for plant genome editing, multi-gene targeting and complex genome editing, base editing and prime editing to achieve more complex genetic engineering. Additionally, new avenues that facilitate fine-tuning plant gene regulation have also been addressed. In spite of the tremendous potential of CRISPR and other gene editing tools, major challenges remain. Some of the challenges are related to the practical advances required for the efficient delivery of CRISPR reagents and for precision genome editing, while others come from government policies and public acceptance. This review will therefore be helpful to gain insights into technological advances, its applications, and future challenges for crop improvement.
Applied Microbiology and Biotechnology, Volume 105, pp 4357-4367; doi:10.1007/s00253-021-11357-0
Acetic acid bacteria (AAB) are a group of Gram-negative and strictly aerobic microorganisms widely used in vinegar industry, especially the species belonging to the genera Acetobacter and Komagataeibacter. The environments inhabited by AAB during the vinegar fermentation, in particular those natural traditional bioprocesses, are complex and dynamically changed, usually accompanied by diverse microorganisms, bacteriophages, and the increasing acetic acid concentration. For this reason, how AAB survive to such harsh niches has always been an interesting research field. Previous omic analyses (e.g., genomics, proteomics, and transcriptomics) have provided abundant clues for the metabolic pathways and bioprocesses indispensable for the acid stress adaptation of AAB. Nevertheless, it is far from fully understanding what factors regulate these modular mechanisms overtly and covertly upon shifting environments. Bacterial toxin-antitoxin systems (TAS), usually consisting of a pair of genes encoding a stable toxin and an unstable antitoxin that is capable of counteracting the toxin, have been uncovered to have a variety of biological functions. Recent studies focusing on the role of TAS in Acetobacter pasteurianus suggest that TAS contribute substantially to the acid stress resistance. In this mini review, we discuss the biological functions of type II TAS in the context of AAB with regard to the acid stress resistance, persister formation and resuscitation, genome stability, and phage immunity. • Type II TAS act as regulators in the acid stress resistance of AAB. • Type II TAS are implicated in the formation of acid-tolerant persister cells in AAB. • Type II TAS are potential factors responsible for phage immunity and genome stability.
Biochemistry (Moscow), Volume 86, pp 449-470; doi:10.1134/s0006297921040064
Bacteriophages or phages are viruses that infect bacterial cells (for the scope of this review we will also consider viruses that infect Archaea). The constant threat of phage infection is a major force that shapes evolution of microbial genomes. To withstand infection, bacteria had evolved numerous strategies to avoid recognition by phages or to directly interfere with phage propagation inside the cell. Classical molecular biology and genetic engineering had been deeply intertwined with the study of phages and host defenses. Nowadays, owing to the rise of phage therapy, broad application of CRISPR-Cas technologies, and development of bioinformatics approaches that facilitate discovery of new systems, phage biology experiences a revival. This review describes variety of strategies employed by microbes to counter phage infection. In the first part defense associated with cell surface, roles of small molecules, and innate immunity systems relying on DNA modification were discussed. The second part focuses on adaptive immunity systems, abortive infection mechanisms, defenses associated with mobile genetic elements, and novel systems discovered in recent years through metagenomic mining.
The Botanical Review pp 1-22; doi:10.1007/s12229-021-09250-6
It’s an evolution of its own kind that a technology changed the interface of biology in such a short expanse of time. Merely a decade ago, scientists reported that the CRISPR-Cas (clustered regularly interspaced short Palindromic repeats-CRISPR associated) system is the part of bacteria and archea’s adaptive immune system which helps in withstanding the attack against invading viruses by acquiring genetic records of invaders to facilitate robust interference upon reinfection. In this Review, we discuss the evolution of CRISPR along the time and recent advances in understanding the vivid mechanism by which Cas proteins respond to foreign nucleic acids and how these systems have been harnessed for precise genome manipulation in plants. With the advancement in this technology, it will become easier to genetically modify the plants for crop improvement.
Metabolic Engineering, Volume 63, pp 141-147; doi:10.1016/j.ymben.2020.12.002
In metabolic engineering, genome editing tools make it much easier to discover and evaluate relevant genes and pathways and construct strains. Clustered regularly interspaced palindromic repeats (CRISPR)-associated (Cas) systems now have become the first choice for genome engineering in many organisms includingindustrially relevant ones. Targeted DNA cleavage by CRISPR-Cas provides variousgenome engineering modes such as indels, replacements, large deletions, knock-in and chromosomal rearrangements, while host-dependent differences in repair pathways need to be considered. The versatility of the CRISPR system has given rise to derivative technologies that complement nuclease-based editing, which causes cytotoxicity especially in microorganisms. Deaminase-mediated base editing installs targeted point mutations with much less toxicity. CRISPRi and CRISPRa can temporarily control gene expression without changing the genomic sequence. Multiplex, combinatorial and large scale editing are made possible by streamlined design and construction of gRNA libraries to further accelerates comprehensive discovery, evaluation and building of metabolic pathways. This review summarizes the technical basis and recent advances in CRISPR-related genome editing tools applied for metabolic engineering purposes, with representative examples of industrially relevant eukaryotic and prokaryotic organisms.
Published: 30 September 2020
CRISPR-Cas adaptive immune systems enable bacteria and archaea to efficiently respond to viral pathogens by creating a genomic record of previous encounters. These systems are broadly distributed across prokaryotic taxa, yet are surprisingly absent in a majority of organisms, suggesting that the benefits of adaptive immunity frequently do not outweigh the costs. Here, combining experiments and models, we show that a delayed immune response which allows viruses to transiently redirect cellular resources to reproduction, which we call “immune lag”, is extremely costly during viral outbreaks, even to completely immune hosts. Critically, the costs of lag are only revealed by examining the non-equilibrium dynamics of a host-virus system occurring immediately after viral challenge. Lag is a basic parameter of microbial defense, relevant to all intracellular, post-infection antiviral defense systems, that has to-date been largely ignored by theoretical and experimental treatments of host-phage systems.