Microbiology

This manuscript details two modified protocols for the isolation of long-stranded or high molecular weight (HMW) DNA from Magnaporthaceae (Ascomycota) fungal mycelium intended for whole genome sequencing. The Cytiva Nucleon PhytoPure and the Macherey-Nagel NucleoBond HMW DNA kits were selected because the former requires lower amounts of starting material and the latter utilizes gentler methods to maximize DNA length, albeit at a higher requirement for input material. The Cytiva Nucleon PhytoPure kit successfully recovered HMW DNA for half of our fungal species by increasing the amount of RNase A treatment and adding in a proteinase K treatment. To reduce the impact of pigmentation development, which occurs toward later stages of culturing, extractions were run in quadruplicate to increase overall DNA concentration. We also adapted the Macherey-Nagel NucleoBond HMW DNA kit for high-quality HMW DNA by grinding the sample to a fine powder, overnight lysis, and splitting the sample before washing the precipitated DNA. For both kits, precipitated DNA was spooled out pre-washing, ensuring a higher percentage of high-integrity long strands. The Macherey-Nagel protocol offers advantages over the first through the utilization of gravity columns that provide gentler treatment, yielding >50% of high-purity DNA strands exceeding 40 kbp. The limitation of this method is the requirement for a large quantity of starting material (1 g). By triaging samples based on the rate of growth relative to the accumulation of secondary metabolites, our methodologies hold promise for yielding reliable and high-quality HMW DNA from a variety of fungal samples, improving sequencing outcomes.

Genome walking, a molecular technique for mining unknown flanking DNAs, has a wide range of uses in life sciences and related areas. Herein, a simple but reliable genome walking protocol named primer extension refractory PCR (PER-PCR) is detailed. This PER-PCR-based protocol uses a set of three walking primers (WPs): primary WP (PWP), secondary WP (SWP), and tertiary WP (TWP). The 15 nt middle region of PWP overlaps the 3' region of SWP/TWP. The 5' regions of the three WPs are completely different from each other. In the low annealing temperature cycle of secondary or tertiary PER-PCR, the short overlap mediates the annealing of the WP to the previous WP site, thus producing a series of single-stranded DNAs (ssDNA). However, the 5' mismatch between the two WPs prevents the template DNA from synthesizing the WP complement at its 3' end. In the next high annealing temperature cycles, the target ssDNA is exponentially amplified because it is defined by both the WP and sequence-specific primer, while non-target ssDNA cannot be amplified as it lacks a binding site for at least one of the primers. Finally, the target DNA becomes the main PER-PCR product. This protocol has been validated by walking two selected genes.

Human astroviruses pose a significant public health threat, especially to children, the elderly, and immunocompromised individuals. Nevertheless, these viruses remain largely understudied, with no approved antivirals or vaccines. This protocol focuses on leveraging reverse genetics (RG) and replicon systems to unravel the biology of MLB genotypes, a key group of neurotropic astroviruses. Using reverse genetics and replicon systems, we identified critical genetic deletions linked to viral attenuation and neurotropism, pushing forward vaccine development. We also uncovered novel replication mechanisms involving ER membrane interactions, opening doors to new antiviral targets. Reverse genetics and replicon systems are essential for advancing our understanding of astrovirus biology, identifying virulence factors, and developing effective treatments and vaccines to combat their growing public health impact.

Tuberculosis (TB) remains the leading cause of human mortality in infectious diseases. Drug-resistant TB, particularly multidrug-resistant TB and extensively drug-resistant TB, poses a pressing clinical and public health challenge. The main causative agents of TB are known as Mycobacterium tuberculosis (MTB), which exhibits a highly complex drug resistance profile. Traditional culture-based phenotypic drug susceptibility testing is time-consuming, and PCR-based assays are restricted to detecting known mutational hotspots. In this study, we present a protocol leveraging high-throughput nanopore sequencing technology in conjunction with multiplex PCR, termed targeted nanopore sequencing, for the identification of MTB and analysis of its drug resistance. Our method for MTB drug resistance assessment offers the benefits of being culture-free, efficient, high-throughput, and highly accurate, which could significantly aid in clinical patient management and the control of TB infections.

Genome walking is a popular molecular technique for accessing unknown flanking DNAs, which has been widely used in biology-related fields. Herein, a simple but accurate genome-walking protocol named partially overlapping primer (POP)-based PCR (POP-PCR) is described. This protocol exploits a POP set of three POPs to mediate genome walking. The three POPs have a 10 nt 3' overlap and 15 nt heterologous 5' regions. Therefore, a POP can partially anneal to the previous POP site only at a relatively low temperature (approximately 50 °C). In primary POP-PCR, the low-temperature (25 °C) cycle allows the primary POP to partially anneal to site(s) of an unknown flank and many sites of the genome, synthesizing many single-stranded DNAs. In the subsequent high-temperature (65 °C) cycle, the target single-stranded DNA is converted into double-stranded DNA by the sequence-specific primer, attributed to the presence of this primer complement, while non-target single-stranded DNA cannot become double-stranded because it lacks a binding site for both primers. As a result, only the target DNA is amplified in the remaining 65 °C cycles. In secondary or tertiary POP-PCR, the 50 °C cycle directs the POP to the previous POP site and synthesizes many single-stranded DNAs. However, as in the primary PCR, only the target DNA can be amplified in the subsequent 65 °C cycles. This POP-PCR protocol has many potential applications, such as screening microbes, identifying transgenic sites, or mining new genetic resources.

PCR-based genome walking is one of the prevalent techniques implemented to acquire unknown flanking genomic DNAs. The worth of genome walking includes but is not limited to cloning full-length genes, mining new genes, and discovering regulatory regions of genes. Therefore, this technique has advanced molecular biology and related fields. However, the PCR amplification specificity of this technique needs to be further improved. Here, a practical protocol based on fork PCR is proposed for genome walking. This PCR uses a fork primer set of three arbitrary primers to execute walking amplification task, where the primary fork primer mediates walking by partially annealing to an unknown flank, and the fork-like structure formed between the three primers participates in inhibiting non-target amplification. In primary fork PCR, the low-annealing temperature (25 °C) cycle allows the primary fork primer to anneal to many sites of the genome, synthesizing a cluster of single-stranded DNAs; the subsequent 65 °C cycle processes the target single-strand into double-strand via the site-specific primer; then, the remaining 65 °C cycles selectively enrich this target DNA. However, any non-target single-stranded DNA formed in the 25 °C cycle cannot be further processed in the following 65 °C cycles because it lacks an exact binding site for any primer. Secondary, or even tertiary nested fork PCR further selectively enriches the target DNA. The practicability of fork PCR was validated by walking three genes in Levilactobacillus brevis CD0817 and one gene in Oryza sativa. The results indicated that the proposed protocol can serve as a supplement to the existing genome walking protocols.

Droplet microfluidic platforms have been broadly used to facilitate DNA transfer in mammalian and bacterial hosts via methods such as transformation, transfection, and conjugation, as introduced in our previous work. Herein, we recapitulate our method for conjugal DNA transfer between Bacillus subtilis strains in a droplet for increased conjugation efficiency and throughput of an otherwise laborious protocol. By co-incubating the donor and recipient strains in droplets, our method confines cells into close proximity allowing for increased cell-to-cell interactions. This methodology is advantageous in its potential to automate and accelerate the genetic modification of undomesticated organisms that may be difficult to cultivate. This device is also designed for modularity and can be integrated into a variety of experimental workflows in which fine-tuning of donor-to-recipient cell ratios, growth rates, and media substrate concentrations may be necessary.

Human babesiosis is a tick-borne disease caused by Babesia pathogens. The disease, which presents with malaria-like symptoms, can be life-threatening, especially in individuals with weakened immune systems and the elderly. The worldwide prevalence of human babesiosis has been gradually rising, prompting alarm among public health experts. In other pathogens, genetic techniques have proven to be valuable tools for conducting functional studies to understand the importance of specific genes in development and pathogenesis as well as to validate novel cellular targets for drug discovery. Genetic manipulation methods have been established for several non-human Babesia and Theileria species and, more recently, have begun to be developed for human Babesia parasites. We have previously reported the development of a method for genetic manipulation of the human pathogen Babesia duncani. This method is based on positive selection using the hDHFR gene as a selectable marker, whose expression is regulated by the ef-1aB promoter, along with homology regions that facilitate integration into the gene of interest through homologous recombination. Herein, we provide a detailed description of the steps needed to implement this strategy in B. duncani to study gene function. It is anticipated that the implementation of this method will significantly improve our understanding of babesiosis and facilitate the development of novel and more effective therapeutic strategies for the treatment of human babesiosis.

The Auxin-inducible degron (AID) system is a genetic tool that induces rapid target protein depletion in an auxin-dependent manner. Recently, two advanced AID systems—the super-sensitive AID and AID 2—were developed using an improved pair of synthetic auxins and mutated TIR1 proteins. In these AID systems, a nanomolar concentration of synthetic auxins is sufficient as a degradation inducer for target proteins. However, despite these advancements, AID systems still require the fusion of an AID tag to the target protein for degradation, potentially affecting its function and stability. To address this limitation, we developed an affinity linker–based super-sensitive AID (AlissAID) system using a single peptide antibody known as a nanobody. In this system, the degradation of GFP- or mCherry-tagged target proteins is induced in a synthetic auxin (5-Ad-IAA)–dependent manner. Here, we introduce a simple method for generating AlissAID strains targeting GFP or mCherry fusion proteins in budding yeasts.

Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) has become the state of the art for mutagenesis in filamentous fungi. Here, we describe a ribonucleoprotein complex (RNP)-mediated CRISPR/Cas9 for mutagenesis in Sporisorium reilianum. The efficiency of the method was tested in vitro with a cleavage assay as well as in vivo with a GFP-expressing S. reilianum strain. We applied this method to generate frameshift- and knock-out mutants in S. reilianum without a resistance marker by using an auto-replicating plasmid for selection. The RNP-mediated CRISPR/Cas9 increased the mutagenesis efficiency, can be applied for all kinds of mutations, and enables a marker-free genome editing in S. reilianum.