RESUMO
Jumbo phages such as Pseudomonas aeruginosa ФKZ have potential as antimicrobials and as a model for uncovering basic phage biology. Both pursuits are currently limited by a lack of genetic engineering tools due to a proteinaceous 'phage nucleus' structure that protects from DNA-targeting CRISPR-Cas tools. To provide reverse-genetics tools for DNA jumbo phages from this family, we combined homologous recombination with an RNA-targeting CRISPR-Cas13a enzyme and used an anti-CRISPR gene (acrVIA1) as a selectable marker. We showed that this process can insert foreign genes, delete genes and add fluorescent tags to genes in the ФKZ genome. Fluorescent tagging of endogenous gp93 revealed that it is ejected with the phage DNA while deletion of the tubulin-like protein PhuZ surprisingly had only a modest impact on phage burst size. Editing of two other phages that resist DNA-targeting CRISPR-Cas systems was also achieved. RNA-targeting Cas13a holds great promise for becoming a universal genetic editing tool for intractable phages, enabling the systematic study of phage genes of unknown function.
Assuntos
Bacteriófagos , Bacteriófagos/genética , Sistemas CRISPR-Cas , Edição de Genes , Engenharia Genética , RNARESUMO
All viruses require strategies to inhibit or evade the immune pathways of cells that they infect. The viruses that infect bacteria, bacteriophages (phages), must avoid immune pathways that target nucleic acids, such as CRISPR-Cas and restriction-modification systems, to replicate efficiently1. Here we show that jumbo phage ΦKZ segregates its DNA from immunity nucleases of its host, Pseudomonas aeruginosa, by constructing a proteinaceous nucleus-like compartment. ΦKZ is resistant to many immunity mechanisms that target DNA in vivo, including two subtypes of CRISPR-Cas3, Cas9, Cas12a and the restriction enzymes HsdRMS and EcoRI. Cas proteins and restriction enzymes are unable to access the phage DNA throughout the infection, but engineering the relocalization of EcoRI inside the compartment enables targeting of the phage and protection of host cells. Moreover, ΦKZ is sensitive to Cas13a-a CRISPR-Cas enzyme that targets RNA-probably owing to phage mRNA localizing to the cytoplasm. Collectively, we propose that Pseudomonas jumbo phages evade a broad spectrum of DNA-targeting nucleases through the assembly of a protein barrier around their genome.
Assuntos
Proteínas Associadas a CRISPR/metabolismo , Fagos de Pseudomonas/genética , Pseudomonas aeruginosa/imunologia , Pseudomonas aeruginosa/virologia , Proteínas Virais/química , Sistemas CRISPR-Cas , DNA Viral/química , Genoma Viral , Fagos de Pseudomonas/químicaRESUMO
How RNA-targeting CRISPR-Cas13 functions as a phage defense system has been mysterious. Recently in Nature, Meeske et al. (2019) demonstrate that Cas13 provides potent immunity to dsDNA phages without cutting their genome. By sensing phage transcripts and destroying RNA nonspecifically to arrest the cell into dormancy, Cas13 provides herd immunity.
Assuntos
Bacteriófagos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , BactériasRESUMO
CRISPR-Cas systems are adaptive immune systems that protect their hosts from predation by bacteriophages (phages) and parasitism by other mobile genetic elements (MGEs). Given the potent nuclease activity of CRISPR effectors, these enzymes must be carefully regulated to minimize toxicity and maximize anti-phage immunity. While attention has been given to the transcriptional regulation of these systems (reviewed in [1]), less consideration has been given to the crucial post-translational processes that govern enzyme activation and inactivation. Here, we review recent findings that describe how Cas nucleases are controlled in diverse systems to provide a robust anti-viral response while limiting auto-immunity. We also draw comparisons to a distinct bacterial immune system, restriction-modification.
Assuntos
Bactérias/genética , Sistemas CRISPR-Cas/genética , Regulação Bacteriana da Expressão Gênica/imunologia , Bactérias/enzimologia , Bactérias/imunologia , Bacteriófagos/genética , Sistemas CRISPR-Cas/imunologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Desoxirribonucleases/genética , Desoxirribonucleases/metabolismoRESUMO
Pre-mRNA splicing is an essential step of eukaryotic gene expression that requires both high efficiency and high fidelity. Prp8 has long been considered the "master regulator" of the spliceosome, the molecular machine that executes pre-mRNA splicing. Cross-linking and structural studies place the RNaseH domain (RH) of Prp8 near the spliceosome's catalytic core and demonstrate that prp8 alleles that map to a 17-aa extension in RH stabilize it in one of two mutually exclusive structures, the biological relevance of which are unknown. We performed an extensive characterization of prp8 alleles that map to this extension and, using in vitro and in vivo reporter assays, show they fall into two functional classes associated with the two structures: those that promote error-prone/efficient splicing and those that promote hyperaccurate/inefficient splicing. Identification of global locations of endogenous splice-site activation by lariat sequencing confirms the fidelity effects seen in our reporter assays. Furthermore, we show that error-prone/efficient RH alleles suppress a prp2 mutant deficient at promoting the first catalytic step of splicing, whereas hyperaccurate/inefficient RH alleles exhibit synthetic sickness. Together our data indicate that prp8 RH alleles link splicing fidelity with catalytic efficiency by biasing the relative stabilities of distinct spliceosome conformations. We hypothesize that the spliceosome "toggles" between such error-prone/efficient and hyperaccurate/inefficient conformations during the splicing cycle to regulate splicing fidelity.