Microbiology

Malaria molecular surveillance has great potential to support national malaria control programs (NMCPs), informing policy for its control and elimination. Here, we present a new three-day workflow for targeted resequencing of markers in 13 resistance-associated genes, histidine rich protein 2 and 3 (hrp2&3), a country (Peru)-specific 28 SNP-barcode for population genetic analysis, and apical membrane antigen 1 (ama1), using Illumina short-read sequencing technology. The assay applies a multiplex PCR approach to amplify all genomic regions of interest in a rapid and easily standardizable procedure and allows simultaneous amplification of a high number of targets at once, therefore having great potential for implementation into routine surveillance practice by NMCPs. The assay can be performed on routinely collected filter paper blood spots and can be easily adapted to different regions to investigate either regional trends or in-country epidemiological changes.

Geobacillus kaustophilus, a thermophilic Gram-positive bacterium, is an attractive host for the development of high-temperature bioprocesses. However, its reluctance against genetic manipulation by standard methodologies hampers its exploitation. Here, we describe a simple methodology in which an artificial DNA segment on the chromosome of Bacillus subtilis can be transferred via pLS20-mediated conjugation resulting in subsequent integration in the genome of G. kaustophilus. Therefore, we have developed a transformation strategy to design an artificial DNA segment on the chromosome of B. subtilis and introduce it into G. kaustophilus. The artificial DNA segment can be freely designed by taking advantage of the plasticity of the B. subtilis genome and combined with the simplicity of pLS20 conjugation transfer. This transformation strategy would adapt to various Gram-positive bacteria other than G. kaustophilus.

Graphical abstract:

Understanding the generation of mutations is fundamental to understanding evolution and genetic disease; however, the rarity of such events makes experimentally identifying them difficult. Mutation accumulation (MA) methods have been widely used. MA lines require serial bottlenecks to fix de novo mutations, followed by whole-genome sequencing. While powerful, this method is not suitable for exploring mutation variation among different genotypes due to its poor scalability with cost and labor. Alternatively, fluctuation assays estimate mutation rate in microorganisms by utilizing a reporter gene, in which Loss-of-function (LOF) mutations can be selected for using drugs toxic to cells containing the WT allele. Traditional fluctuation assays can estimate mutation rates but not their base change compositions. Here, we describe a new protocol that adapts traditional fluctuation assay using CAN1 reporter gene in Saccharomyces cerevisiae, followed by pooled sequencing methods, to identify both the rate and spectra of mutations in different strain backgrounds.

DNA methylation is a conserved chemical modification, by which methyl groups are added to the cytosine of DNA molecules. Methylation can influence gene expression without changing the sequence of a particular gene. This epigenetic effect is an intriguing phenomenon that has puzzled biologists for years. By probing the temporal and spatial patterns of DNA methylation in genomes, it is possible to learn about the biological role of cytosine methylation, as well as its involvement in gene regulation and transposon silencing. Advances in whole-genome sequencing have led to the widespread adoption of methods that examine genome-wide patterns of DNA methylation. Achieving sufficient sequencing depth in these types of experiments is costly, particularly for pilot studies in organisms with large genome sizes, or incomplete reference genomes. To overcome this issue, assays to determine site-specific DNA methylation can be used. Although often used, these assays are rarely described in detail. Here, we describe a pipeline that applies traditional TA cloning, Sanger sequencing, and online tools to examine DNA methylation. We provide an example of how to use this protocol to examine the pattern of DNA methylation at a specific transposable element in maize.

Phytophthora sojae is a model species for the study of plant pathogenic oomycetes. The initial research on gene function using Phytophthora was mainly based on gene silencing technology. Recently, the CRISPR/Cas9-mediated genome editing technology was successfully established in P. sojae and widely used in oomycetes. In this protocol, we describe the operating procedures for the use of CRISPR/Cas9-based genome editing technology and PEG-mediated stable transformation of P. sojae protoplasts. Two plasmids were co-transformed into P. sojae: pYF515 expressing Cas9 and the single guide RNA, and the homologous replacement vector of the candidate gene. Finally, the ORF of candidate gene were replaced with the ORF of the entire hygromycin B phosphotransferase gene (HPH), to achieve precise knockout.

Directed evolution is a powerful approach to obtain genetically-encoded sought-for traits. Compared to the prolonged adaptation regimes to mutations occurring under natural selection, directed evolution unlocks rapid screening and selection of mutants with improved traits from vast mutated sequence spaces. Many systems have been developed to search variant landscapes based on ex vivo or in vivo mutagenesis, to identify and select new-to-nature and optimized properties in biomolecules. Yet, the majority of such systems rely on tedious iterations of library preparation, propagation, and selection steps. Furthermore, among the relatively few in vivo directed evolution systems developed to mitigate handling of repetitive ex vivo steps, directed evolution of DNA is the standard approach. Here, we present the protocol for designing the transfer of genetic information from evolving RNA donors to DNA in baker’s yeast, using CRISPR- and RNA-assisted in vivo directed evolution (CRAIDE). We use mutant T7 RNA polymerase to introduce mutations in RNA donors, while incorporation into DNA is directed by CRISPR/Cas9. As such, CRAIDE offers an opportunity to study fundamental questions, such as RNA’s contribution to the evolution of DNA-based life on Earth.

Graphic abstract:

CRISPR- and RNA-assisted in vivo directed evolution (CRAIDE).

At the end of about 80% of the operon in Escherichia coli, translation termination decouples transcription, leading to Rho-dependent transcription termination (RDT). However, no in vitro or in vivo assay system has proven to be good enough to see the 3’ end of the mRNA generated by RDT. Here, we present a cell-free assay system that could provide detailed information on the 3’ end of a transcript RNA generated by RDT. Our protocol shows how to extract transcript RNA generated by transcription reactions from a cell-free extract, followed by an RNA oligomer ligation to the 3’ end of a transcript RNA of interest. The 3' end of the RNA is amplified using RT-PCR. Its genetic location can be determined using a gene-specific primer extension reaction. The 3’ ends of mRNA can be visualized and quantified by polyacrylamide gel electrophoresis. One significant advantage of a cell-free assay system is that factors involved in the generation of the 3' end, such as proteins and sRNA, can be directly assayed by exogenously adding factor(s) to the reaction.

Graphic abstract:

An illustration of the experimental methodology.

The engineering of poxvirus genomes is fundamental to primary and applied virology research. Indeed, recombinant poxviruses form the basis for many novel vaccines and virotherapies but producing and purifying these viruses can be arduous. In recent years, CRISPR/Cas9 has become the favoured approach for genome manipulation due to its speed and high success rate. However, recent data suggests poxvirus genomes are not repaired well following Cas9 cleavage. As a result, CRISPR/Cas9 is inefficient as an editing tool, but very effective as a programmable selection agent. Here, we describe protocols for the generation and enrichment of recombinant vaccinia viruses using targeted Cas9 as a selection tool. This novel use of Cas9 is a simple addition to current homologous recombination-based methods that are widespread in the field, facilitating implementation in laboratories already working with poxviruses. This is also the first method that allows for isolation of new vaccinia viruses in less than a fortnight, without the need to incorporate a marker gene or manipulation of large poxvirus genomes in vitro and reactivation with helper viruses. Whilst this protocol describes applications for laboratory strains of vaccinia virus, it should be readily adaptable to other poxviruses.

Graphic abstract:

Pipeline for Cas9 selection of recombinant poxviruses.

The ongoing COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As this virus is classified as a biosafety level-3 (BSL-3) agent, the development of countermeasures and basic research methods is logistically difficult. Recently, using reverse genetics, we developed a BSL-2 cell culture system for production of transcription- and replication-component virus-like-particles (trVLPs) by genetic transcomplementation. The system consists of two parts: SARS-CoV-2 GFP/ΔN genomic RNA, in which the nucleocapsid (N) gene, a critical gene for virion packaging, is replaced by a GFP reporter gene; and a packaging cell line for ectopic expression of N (Caco-2-N). The complete viral life cycle can be recapitulated and confined to Caco-2-N cells, with GFP positivity serving as a surrogate readout for viral infection. In addition, we utilized an intein-mediated protein splicing technique to split the N gene into two independent vectors and generated the Caco-2-N^intein cells as a packaging cell line to further enhance the security of this cell culture model. Altogether, this system provides for a safe and convenient method to produce trVLPs in BSL-2 laboratories. These trVLPs can be modified to incorporate desired mutations, permitting high-throughput screening of antiviral compounds and evaluation of neutralizing antibodies. This protocol describes the details of the trVLP cell culture model to make SARS-CoV-2 research more readily accessible.

The fission yeast Schizosaccharomyces pombe is an excellent genetically tractable model organism used in the study of conserved eukaryotic cellular biology. One genetic tool in the assessment of gene function is the in vivo overexpression of proteins. Existing overexpression tools have limitations of induction kinetics, dynamic range, and/or system-wide changes due to the induction conditions or inducer. Here, I describe the methodology for the use of a plasmid-based long non-coding RNA (lncRNA)-regulated overexpression system that is induced by the addition of thiamine. This system, termed the pTIN-system (thiamine inducible), utilizes the fast repression kinetics of the thiamine-regulated nmt1⁺ promoter integrated with the lncRNA regulated tgp1⁺ promoter. The advantages of the pTIN-system are rapid induction kinetics of gene expression, broad dynamic range, and tunable expression.