Evolution of Restriction-Modification Systems Consisting of One Restriction Endonuclease and Two DNA Methyltransferases.

Some restriction-modification systems contain two DNA methyltransferases. In the present work, we have classified such systems according to the families of catalytic domains present in the restriction endonucleases and both DNA methyltransferases. Evolution of the restriction-modification systems containing an endonuclease with a NOV_C family domain and two DNA methyltransferases, both with DNA_methylase family domains, was investigated in detail. Phylogenetic tree of DNA methyltransferases from the systems of this class consists of two clades of the same size. Two DNA methyltransferases of each restriction-modification system of this class belong to the different clades. This indicates independent evolution of the two methyltransferases. We detected multiple cross-species horizontal transfers of the systems as a whole, as well as the cases of gene transfer between the systems.

[Anti-Restriction Activity of ArdB Protein against EcoAI Endonuclease].

ArdB proteins are known to inhibit the activity of the type I restriction-modification (RM-I) system, in particular EcoKI (IA family). The mechanism of ArdB's activity still remains unknown; the spectrum of targets inhibited has been poorly studied. In this work, it was shown that the presence of the ardB gene from the R64 plasmid could suppress the activity of EcoAI endonuclease (IB family) in Escherichia coli TG1 cells. Due to the absence of specificity of ArdB to a certain RM-I system (it inhibits both the IA- and IB-family), it can be assumed that the mechanism of the anti-restriction activity of this protein does not depend on the sequence DNA at the recognition site nor the structure of the restriction enzyme of the RM-I systems.

Restriction endonuclease cleavage of phage DNA enables resuscitation from Cas13-induced bacterial dormancy.

Type VI CRISPR systems protect against phage infection using the RNA-guided nuclease Cas13 to recognize viral messenger RNA. Upon target recognition, Cas13 cleaves phage and host transcripts non-specifically, leading to cell dormancy that is incompatible with phage propagation. However, whether and how infected cells recover from dormancy is unclear. Here we show that type VI CRISPR and DNA-cleaving restriction-modification (RM) systems frequently co-occur and synergize to clear phage infections and resuscitate cells. In the natural type VI CRISPR host Listeria seeligeri, we show that RM cleaves the phage genome, thus removing the source of phage transcripts and enabling cells to recover from Cas13-induced cellular dormancy. We find that phage infections are neutralized more effectively when Cas13 and RM systems operate together. Our work reveals that type VI CRISPR immunity is cell-autonomous and non-abortive when paired with RM, and hints at other synergistic roles for the diverse host-directed immune systems in bacteria.

Computational Protocol for DNA Methylation Profiling in Plants Using Restriction Enzyme-Based Genome Reduction.

Epigenetics can be described as heritable phenotype changes that do not involve alterations in the underlying DNA sequence. Having widespread implications in fundamental biological phenomena, there is an increased interest in characterizing epigenetic modifications and studying their functional implications. DNA methylation, particularly 5-methylcytosine (5mC), stands out as the most studied epigenetic mark and several methodologies have been created to investigate it. With the development of next-generation sequencing technologies, several approaches to DNA methylation profiling were conceived, with differences in resolution and genomic scope. Besides the gold standard whole-genome bisulfite sequencing, which is costly for population-scale studies, genomic reduced representation methods emerged as viable alternatives to investigate methylation loci. Whole-genome bisulfite sequencing provides single-base methylation resolution but is costly for population-scale studies. Genomic reduction methods emerged as viable alternatives to investigate a fraction of methylated loci. One of such approaches uses double digestion with the restriction enzymes PstI and one of the isoschizomers, MspI and HpaII, with differential sensitivity to 5mC at the restriction site. Statistical comparison of sequencing reads counts obtained from the two libraries for each sample (PstI-MspI and PstI-HpaII) is used to infer the methylation status of thousands of cytosines. Here, we describe a general overview of the technique and a computational protocol to process the generated data to provide a medium-scale inventory of methylated sites in plant genomes. The software is available at https://github.com/wendelljpereira/DArTseqMet .

ORE-Seq: Genome-Wide Absolute Occupancy Measurement by Restriction Enzyme Accessibilities.

Digestion with restriction enzymes is a classical approach for probing DNA accessibility in chromatin. It allows to monitor both the cut and the uncut fraction and thereby the determination of accessibility or occupancy (= 1 - accessibility) in absolute terms as the percentage of cut or uncut molecules, respectively, out of all molecules. The protocol presented here takes this classical approach to the genome-wide level. After exhaustive restriction enzyme digestion of chromatin, DNA is purified, sheared, and converted into libraries for high-throughput sequencing. Bioinformatic analysis counts uncut DNA fragments as well as DNA ends generated by restriction enzyme digest and derives thereof the fraction of accessible DNA. This straightforward principle is technically challenged as preparation and sequencing of the libraries leads to biased scoring of DNA fragments. Our protocol includes two orthogonal approaches to correct for this bias, the "corrected cut-uncut" and the "cut-all cut" method, so that accurate measurements of absolute accessibility or occupancy at restriction sites throughout a genome are possible. The protocol is presented for the example of S. cerevisiae chromatin but may be adapted for any other species.

A novel, ultrafast, ultrasensitive diagnosis platform for the detection of SARS-COV-2 using restriction endonuclease-mediated reverse transcription multiple cross displacement amplification.

Coronavirus disease 2019 (COVID-19) is a highly infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-COV-2). Though many methods have been used for detecting SARS-COV-2, development of an ultrafast and highly sensitive detection strategy to screen and/or diagnose suspected cases in the population, especially early-stage patients with low viral load, is significant for the prevention and treatment of COVID-19. In this study, a novel restriction endonuclease-mediated reverse transcription multiple cross displacement amplification (MCDA) combined with real-time fluorescence analysis (rRT-MCDA) was successfully established and performed to diagnose COVID-19 infection (COVID-19 rRT-MCDA). Two sets of specific SARS-COV-2 rRT-MCDA primers targeting opening reading frame 1a/b (ORF1ab) and nucleoprotein (NP) genes were designed and modified according to the reaction mechanism. The SARS-COV-2 rRT-MCDA test was optimized and evaluated using various pathogens and clinical samples. The optimal reaction condition of SARS-COV-2 rRT-MCDA assay was 65°C for 36 min. The SARS-COV-2 rRT-MCDA limit of detection (LoD) was 6.8 copies per reaction. Meanwhile, the specificity of SARS-COV-2 rRT-MCDA assay was 100%, and there was no cross-reaction with nucleic acids of other pathogens. In addition, the whole detection process of SARS-COV-2 rRT-MCDA, containing the RNA template processing (15 min) and real-time amplification (36 min), can be accomplished within 1 h. The SARS-COV-2 rRT-MCDA test established in the current report is a novel, ultrafast, ultrasensitive, and highly specific detection method, which can be performed as a valuable screening and/or diagnostic tool for COVID-19 in clinical application.

Cells with stochastically increased methyltransferase to restriction endonuclease ratio provide an entry for bacteriophage into protected cell population.

The action of Type II restriction-modification (RM) systems depends on restriction endonuclease (REase), which cleaves foreign DNA at specific sites, and methyltransferase (MTase), which protects host genome from restriction by methylating the same sites. We here show that protection from phage infection increases as the copy number of plasmids carrying the Type II RM Esp1396I system is increased. However, since increased plasmid copy number leads to both increased absolute intracellular RM enzyme levels and to a decreased MTase/REase ratio, it is impossible to determine which factor determines resistance/susceptibility to infection. By controlled expression of individual Esp1396I MTase or REase genes in cells carrying the Esp1396I system, we show that a shift in the MTase to REase ratio caused by overproduction of MTase or REase leads, respectively, to decreased or increased protection from infection. Consistently, due to stochastic variation of MTase and REase amount in individual cells, bacterial cells that are productively infected by bacteriophage have significantly higher MTase to REase ratios than cells that ward off the infection. Our results suggest that cells with transiently increased MTase to REase ratio at the time of infection serve as entry points for unmodified phage DNA into protected bacterial populations.

A User's Guide to Golden Gate Cloning Methods and Standards.

The continual demand for specialized molecular cloning techniques that suit a broad range of applications has driven the development of many different cloning strategies. One method that has gained significant traction is Golden Gate assembly, which achieves hierarchical assembly of DNA parts by utilizing Type IIS restriction enzymes to produce user-specified sticky ends on cut DNA fragments. This technique has been modularized and standardized, and includes different subfamilies of methods, the most widely adopted of which are the MoClo and Golden Braid standards. Moreover, specialized toolboxes tailored to specific applications or organisms are also available. Still, the quantity and range of assembly methods can constitute a barrier to adoption for new users, and even experienced scientists might find it difficult to discern which tools are best suited toward their goals. In this review, we provide a beginner-friendly guide to Golden Gate assembly, compare the different available standards, and detail the specific features and quirks of commonly used toolboxes. We also provide an update on the state-of-the-art in Golden Gate technology, discussing recent advances and challenges to inform existing users and promote standard practices.

New Platform for Screening Genetic Libraries at Elevated Temperatures: Biological and Genomic Information and Genetic Tools of Geobacillus thermodenitrificans K1041.

Geobacillus thermodenitrificans K1041 is an unusual thermophile that is highly transformable via electroporation, making it a promising host for screening genetic libraries at elevated temperatures. In this study, we determined its biological properties, draft genome sequence, and effective vectors and also optimized the electroporation procedures in an effort to enhance its utilization. The organism exhibited swarming motility but not detectable endospore formation, and growth was rapid at 60°C under neutral and relatively low-salt conditions. Although the cells showed negligible acceptance of shuttle plasmids from general strains of Escherichia coli, methylation-controlled plasmids from dam mutant strains were efficiently accepted, suggesting circumvention of a restriction-modification system in G. thermodenitrificans K1041. We optimized the electroporation procedure to achieve efficiencies of 103 to 105 CFU/µg for five types of plasmids, which exhibited the different copy numbers and segregational stabilities in G. thermodenitrificans K1041. Some sets of plasmids were compatible. Moreover, we observed substantial plasmid-directed production of heterologous proteins in the intracellular or extracellular environments. Our successful construction of a library of promoter mutants using K1041 cells as hosts and subsequent screening at elevated temperatures to identify improved promoters revealed that G. thermodenitrificans K1041 was practical as a library host. The draft genomic sequence of the organism contained 3,384 coding genes, including resA and mcrB genes, which are involved in restriction-modification systems. Further examination revealed that in-frame deletions of resA increased transformation efficiencies, but mcrB deletion had no effect. The ΔresA mutant exhibited transformation efficiencies of >105 CFU/µg for some plasmids. IMPORTANCE Geobacillus thermodenitrificans K1041 has yet to be fully characterized. Although it is transformable via electroporation, it rarely accepts Escherichia coli-derived plasmids. This study clarified the biological and genomic properties of G. thermodenitrificans K1041. Additionally, we developed an electroporation procedure resulting in efficient acceptance of E. coli-derived plasmids. This procedure produced transformants using small amounts of plasmids immediately after the ligation reaction. Thus, G. thermodenitrificans K1041 was identified as a host for screening promoter mutants at elevated temperatures. Furthermore, because this strain efficiently produced heterologous proteins, it could serve as a host for screening thermostable proteins encoded in random mutant libraries or metagenomes. We also generated a ΔresA mutant that exhibited transformation efficiencies of >105 CFU/µg, which were highest in cases of electroporation-based transformation of Geobacillus spp. with E. coli-derived plasmids. Our findings provide a new platform for screening diverse genetic libraries at elevated temperatures.

Sensitive lateral flow assay for bisulfite-free DNA methylation detection based on the restriction endonuclease GlaI and rolling circle amplification.

The detection of DNA methylation with high sensitivity and specificity is important for the early diagnosis of many human diseases, including cancers. Here, we integrated the high specificity of the methylation-dependent restriction endonuclease GlaI for methylation-dependent digestion and the high amplification efficiency of rolling circle amplification (RCA) for the detection of GlaI digestion products. GlaI can only digest a methylated template, leading to the generation of digestion products with specific ends. The specific digestion product can then be ligated to a ligation mediator with a dumbbell structure to generate a complete circular template for further RCA, and the final RCA amplicon can be detected using lateral flow detection (LFD) with the naked eye. The specificity of Gla-RCA not only depends on the specific methylation digestion of GlaI, but only the ligation process of RCA amplification. As a proof of principle, the sensitivity of GlaI-RCA assay was applied to methylated Septin 9 and showed a sensitivity of approximately 1% (50 copies of methylated template per reaction) and no cross-reactivity with 5000 copies of unmethylated DNA used as background. The application of GlaI-RCA was also evaluated with colorectal cancer tissue samples and showed great accordance with standard bisulfite sequencing. A bisulfite-free and LFD-based DNA methylation detection was successfully developed, promising high specificity and rapid visual detection and having a great potential to become a robust tool for DNA methylations analysis.

[Specific amplification of plasma exosome miRNA in cancer patients for construction of cDNA library].

Plasma exosome microRNAs (miRNAs) are closely related with the occurrence, diagnosis, and treatment of cancers. However, the underlying molecular mechanisms remain unclear. We herein investigated the solution for tackling the unspecific amplification of plasma exosome microRNAs from cancer patients during the construction of its cDNA library. For the restriction enzyme digesting method, the primers were degraded by exonuclease T (EXOT) and phi29 DNA polymerase. For the magnetic bead separation method, the templates and primers were separated through the DNA binding beads. The separation effects of magnetic beads were detected by agarose gel electrophoresis and modified polyacrylamide gel electrophoresis. The levels of plasma exosome miRNAs from cancer patients and various primers were assayed by RT-qPCR. The results indicated that the unspecific amplification stemmed from USR5SR. EXOT and phi29 DNA polymerase could degrade USR5SR, but the templates were also degraded simultaneously. Regarding the magnetic bead separation method, the best effect was achieved via precipitation of primer fragments by 9% PEG and precipitation of templates by 15% PEG. In conclusion, the magnetic bead separation method efficiently circumvented the unspecific amplification during the construction of cDNA library, and therefore led to the successful construction of cDNA library from plasma exosome miRNA of cancer patients and 293T cells.

Application of Restriction Free (RF) Cloning in Circular Permutation.

The restriction free (RF) cloning has emerged as one of the highly efficient techniques in the area of genetic engineering. RF cloning has wide range of applications in plasmid DNA manipulation including cloning of a single gene, simultaneous assembly of multiple DNA fragments, and mutagenesis from single to multiple simultaneous alterations of a target DNA. Recently, we have developed a new technique of circular permutation using RF cloning. Circular permutation is widely used to investigate the mechanisms of protein folding and function. Previously, restriction enzyme based cloning was used to introduce circular permutation. Our RF cloning method made the protocol faster and more cost-effective. In this chapter, we describe a step-by-step protocol for generating circular permutants using RF methodology.

Population Genomics Analysis with RAD, Reprised: Stacks 2.

Restriction enzymes have been one of the primary tools in the population genetics toolkit for 50 years, being coupled with each new generation of technology to provide a more detailed view into the genetics of natural populations. Restriction site-Associated DNA protocols, which joined enzymes with short-read sequencing technology, have democratized the field of population genomics, providing a means to assay the underlying alleles in scores of populations. More than 10 years on, the technique has been widely applied across the tree of life and served as the basis for many different analysis techniques. Here, we provide a detailed protocol to conduct a RAD analysis from experimental design to de novo analysis-including parameter optimization-as well as reference-based analysis, all in Stacks version 2, which is designed to work with paired-end reads to assemble RAD loci up to 1000 nucleotides in length. The protocol focuses on major points of friction in the molecular approaches and downstream analysis, with special attention given to validating experimental analyses. Finally, the protocol provides several points of departure for further analysis.

PlasmidMaker is a versatile, automated, and high throughput end-to-end platform for plasmid construction.

Plasmids are used extensively in basic and applied biology. However, design and construction of plasmids, specifically the ones carrying complex genetic information, remains one of the most time-consuming, labor-intensive, and rate-limiting steps in performing sophisticated biological experiments. Here, we report the development of a versatile, robust, automated end-to-end platform named PlasmidMaker that allows error-free construction of plasmids with virtually any sequences in a high throughput manner. This platform consists of a most versatile DNA assembly method using Pyrococcus furiosus Argonaute (PfAgo)-based artificial restriction enzymes, a user-friendly frontend for plasmid design, and a backend that streamlines the workflow and integration with a robotic system. As a proof of concept, we used this platform to generate 101 plasmids from six different species ranging from 5 to 18 kb in size from up to 11 DNA fragments. PlasmidMaker should greatly expand the potential of synthetic biology.

A mobile restriction-modification system provides phage defence and resolves an epigenetic conflict with an antagonistic endonuclease.

Epigenetic DNA methylation plays an important role in bacteria by influencing gene expression and allowing discrimination between self-DNA and intruders such as phages and plasmids. Restriction-modification (RM) systems use a methyltransferase (MTase) to modify a specific sequence motif, thus protecting host DNA from cleavage by a cognate restriction endonuclease (REase) while leaving invading DNA vulnerable. Other REases occur solitarily and cleave methylated DNA. REases and RM systems are frequently mobile, influencing horizontal gene transfer by altering the compatibility of the host for foreign DNA uptake. However, whether mobile defence systems affect pre-existing host defences remains obscure. Here, we reveal an epigenetic conflict between an RM system (PcaRCI) and a methylation-dependent REase (PcaRCII) in the plant pathogen Pectobacterium carotovorum RC5297. The PcaRCI RM system provides potent protection against unmethylated plasmids and phages, but its methylation motif is targeted by the methylation-dependent PcaRCII. This potentially lethal co-existence is enabled through epigenetic silencing of the PcaRCII-encoding gene via promoter methylation by the PcaRCI MTase. Comparative genome analyses suggest that the PcaRCII-encoding gene was already present and was silenced upon establishment of the PcaRCI system. These findings provide a striking example for selfishness of RM systems and intracellular competition between different defences.

Assessment of genetic variation in Apis mellifera jemenitica (Hymenoptera: Apidae) based on mitochondrial Cytochrome Oxidase Subunit II and III.

Morphometric and genetic characterization of many Apis mellifera subspecies are well-documented. A. m. jemenetica occurs naturally in Africa and Asia. In this study, genetic variation of mitochondrial Cytochrome Oxidase II (COII) and III (COIII) were analysed in 133 specimens of the endemic honeybee colonies within Saudi Arabia. The COII gene sequence length was 684 bp comprising nine synonymous (1.3%) and two non-synonymous single nucleotide polymorphisms (SNPs) (0.87%). Five variants of COII were not previously documented, one variant (MT755968) showed an extra restriction site when subjected to type II restriction endonuclease from Arthrobacter protophormiae (Apol) or to Haemophilus influenzae Rf (Hinf1). Changes in COII sequence separated samples into three haplogroups. Whereas, COIII gene sequence length was 780 bp, including 18 synonymous and five non-synonymous SNPs. Furthermore, variation in COII sequence was more informative based on restriction profiles and on amino acid changes compared with COIII gene sequence. Variants of COIII showed identical restriction sites when subjected to type II restriction endonuclease from Deinococcus radiophilus (DraI), and revealed high similarity to African subspecies. Results of this study are very useful in understanding genetic diversity and characterization of A. mellifera subspecies.

Cleavage of viral DNA by restriction endonucleases stimulates the type II CRISPR-Cas immune response.

Prokaryotic organisms have developed multiple defense systems against phages; however, little is known about whether and how these interact with each other. Here, we studied the connection between two of the most prominent prokaryotic immune systems: restriction-modification and CRISPR. While both systems employ enzymes that cleave a specific DNA sequence of the invader, CRISPR nucleases are programmed with phage-derived spacer sequences, which are integrated into the CRISPR locus upon infection. We found that restriction endonucleases provide a short-term defense, which is rapidly overcome through methylation of the phage genome. In a small fraction of the cells, however, restriction results in the acquisition of spacer sequences from the cleavage site, which mediates a robust type II-A CRISPR-Cas immune response against the methylated phage. This mechanism is reminiscent of eukaryotic immunity in which the innate response offers a first temporary line of defense and also activates a second and more robust adaptive response.

Restriction enzyme selection dictates detection range sensitivity in chromatin conformation capture-based variant-to-gene mapping approaches.

Promoter-focused chromatin conformation techniques directly detect interactions between gene promoters and distal genomic sequences, providing structural information relevant to gene regulation without the excessive non-genic architectural data generated by full-scale Hi-C. 3D promoter 'interactome' maps are crucial for understanding how epigenomic features such as histone modifications and open chromatin, or genetic variants identified in genome-wide association studies (GWAS), contribute to biological function. However, variation in sensitivity between such promoter-focused methods, principally due to restriction enzyme selection, has not been systematically assessed. Here, we performed a head-to-head comparison of promoter capture datasets using 4 cutters (DpnII or MboI) versus the 6 cutter HindIII from the same five cell types. While HindIII generally produces a higher signal-to-noise ratio for significant interactions in comparison to 4-cutters, we show that DpnII/MboI detects more proximal interactions and shows little overlap with the HindIII detection range. Promoter-interacting genomic regions mapped by 4-cutters are more enriched for regulatory features and disease-associated genetic variation than 6-cutters maps, suggesting that high-resolution maps better capture gene regulatory architectures than do lower resolution approaches.

Rapid identification of methylase specificity (RIMS-seq) jointly identifies methylated motifs and generates shotgun sequencing of bacterial genomes.

DNA methylation is widespread amongst eukaryotes and prokaryotes to modulate gene expression and confer viral resistance. 5-Methylcytosine (m5C) methylation has been described in genomes of a large fraction of bacterial species as part of restriction-modification systems, each composed of a methyltransferase and cognate restriction enzyme. Methylases are site-specific and target sequences vary across organisms. High-throughput methods, such as bisulfite-sequencing can identify m5C at base resolution but require specialized library preparations and single molecule, real-time (SMRT) sequencing usually misses m5C. Here, we present a new method called RIMS-seq (rapid identification of methylase specificity) to simultaneously sequence bacterial genomes and determine m5C methylase specificities using a simple experimental protocol that closely resembles the DNA-seq protocol for Illumina. Importantly, the resulting sequencing quality is identical to DNA-seq, enabling RIMS-seq to substitute standard sequencing of bacterial genomes. Applied to bacteria and synthetic mixed communities, RIMS-seq reveals new methylase specificities, supporting routine study of m5C methylation while sequencing new genomes.

A systematic method for DNA fragment amplification and sequencing based on DNA indexing technology. Protocol and technical considerations.

DNA indexing is based on a presynthesized library of oligonucleotide adaptors (256 in total), named indexers, and type-IIS restriction endonucleases. It enables amplification and direct analysis of large DNA fragments with low overall redundancy and without subcloning. Here, we describe a detailed protocol for PCR-based amplification of DNA fragments followed by DNA sequencing by indexer walking and provide helpful hints on its practical use. The proposed protocol can be applied to the sequencing of plasmids, cDNA clones, and longer DNA fragments. It can also be used for gap filling at the final stage of genome sequencing projects.