Global mpox lineage discovery and rapid outbreak tracking with nanopore sequencing.

Insufficient tracking of virus introduction, spread, and new lineage emergence for the human monkeypox (mpox) virus 1 (hMPXV1) outbreak of 2022 hindered epidemiological studies and public health response. hMPXV1 mutations accumulated unexpectedly faster than predicted. Thus, new variants with altered pathogenicity could emerge and spread without early detection. Whole genome sequencing addresses this gap when implemented but requires widely accessible and standardized methodologies to be effective both regionally and globally. Here we developed a rapid nanopore whole genome sequencing method complete with working protocols, from DNA extraction to phylogenetic analysis tools. Using this method, we sequenced 84 complete hMPXV1 genomes from Illinois, a Midwestern region of the United States, spanning the first few months of the outbreak. The resulting five-fold increase in hMPXV1 genomes from this region established two previously unnamed global lineages, several mutational profiles not seen elsewhere, multiple separate introductions of the virus into the region, and the likely emergence and spread of new lineages from within this region. These results demonstrate that a dearth of genomic sequencing of hMPXV1 slowed our understanding and response to the mpox outbreak. This accessible nanopore sequencing approach makes near real-time mpox tracking and rapid lineage discovery straightforward and creates a blueprint for how to deploy nanopore sequencing for genomic surveillance of diverse viruses and future outbreaks.

Binding to the conserved and stably folded guide RNA pseudoknot induces Cas12a conformational changes during ribonucleoprotein assembly.

Ribonucleoproteins (RNPs) comprise one or more RNA and protein molecules that interact to form a stable complex, which commonly involves conformational changes in the more flexible RNA components. Here, we propose that Cas12a RNP assembly with its cognate CRISPR RNA (crRNA) guide instead proceeds primarily through Cas12a conformational changes during binding to more stable, prefolded crRNA 5' pseudoknot handles. Phylogenetic reconstructions and sequence and structure alignments revealed that the Cas12a proteins are divergent in sequence and structure while the crRNA 5' repeat region, which folds into a pseudoknot and anchors binding to Cas12a, is highly conserved. Molecular dynamics simulations of three Cas12a proteins and their cognate guides revealed substantial flexibility for unbound apo-Cas12a. In contrast, crRNA 5' pseudoknots were predicted to be stable and independently folded. Limited trypsin hydrolysis, differential scanning fluorimetry, thermal denaturation, and CD analyses supported conformational changes of Cas12a during RNP assembly and an independently folded crRNA 5' pseudoknot. This RNP assembly mechanism may be rationalized by evolutionary pressure to conserve CRISPR loci repeat sequence, and therefore guide RNA structure, to maintain function across all phases of the CRISPR defense mechanism.

Efficient Cloning and Sequence Validation of Repetitive and High GC-Content Short Hairpin RNAs.

Short hairpin RNAs, or short hairpin RNAs (shRNAs), are a proven tool for gene knockdown and a promising therapeutic approach for suppression of disease-associated genes. The efficient preparation of shRNA-expressing vectors can sometimes become a bottleneck due to the complexity of shRNA hairpin sequence and structure, especially for repetitive or high GC-content targets. Here, we present improved shRNA cloning and validation methods that enabled efficient and rapid cloning of several shRNAs targeting disease-associated repeat expansions, including GGGGCC, CAG, CTG, CCTG, and CGG into modified pLKO.1 vectors. Improvements included shRNA insert design and preparation, recombination-based cloning, and sequencing-based validation that included Sanger and nanopore long-read sequencing. This improved method should enable practical, efficient cloning of nearly any shRNA sequence.

High throughput nanopore sequencing of SARS-CoV-2 viral genomes from patient samples.

In late 2019, a novel coronavirus began spreading in Wuhan, China, causing a potentially lethal respiratory viral infection. By early 2020, the novel coronavirus, called SARS-CoV-2, had spread globally, causing the COVID-19 pandemic. The infection and mutation rates of SARS-CoV-2 make it amenable to tracking introduction, spread and evolution by viral genome sequencing. Efforts to develop effective public health policies, therapeutics, or vaccines to treat or prevent COVID-19 are also expected to benefit from tracking mutations of the SARS-CoV-2 virus. Here we describe a set of comprehensive working protocols, from viral RNA extraction to analysis using established visualization tools, for high throughput sequencing of SARS-CoV-2 viral genomes using a MinION instrument. This set of protocols should serve as a reliable "how-to" reference for generating quality SARS-CoV-2 genome sequences with ARTIC primer sets and long-read nanopore sequencing technology. In addition, many of the preparation, quality control, and analysis steps will be generally applicable to other sequencing platforms.

Label-free Electrochemical Detection of CGG Repeats on Inkjet Printable 2D Layers of MoS2.

Flexible and ultrasensitive biosensing platforms capable of detecting a large number of trinucleotide repeats (TNRs) are crucial for future technology development needed to combat a variety of genetic disorders. For example, trinucleotide CGG repeat expansions in the FMR1 gene can cause Fragile X syndrome (FXS) and Fragile X-associated tremor/ataxia syndrome (FXTAS). Current state-of-the-art technologies to detect repeat sequences are expensive, while relying on complicated procedures, and prone to false negatives. We reasoned that two-dimensional (2D) molybdenum sulfide (MoS2) surfaces may be useful for label-free electrochemical detection of CGG repeats due to its high affinity for guanine bases. Here, we developed a low-cost and sensitive wax-on-plastic electrochemical sensor using 2D MoS2 ink for the detection of CGG repeats. The ink containing few-layered MoS2 nanosheets was prepared and characterized using optical, electrical, electrochemical, and electron microscopic methods. The devices were characterized by electron microscopic and electrochemical methods. Repetitive CGG DNA was adsorbed on a MoS2 surface in a high cationic strength environment and the electrocatalytic current of the CGG/MoS2 interface was recorded using a soluble Fe(CN)6-3/-4 redox probe by differential pulse voltammetry (DPV). The dynamic range for the detection of prehybridized duplexes ranged from 1 aM to 100 nM with a 3.0 aM limit of detection. A detection range of 100 fM to 1 nM was recorded for surface hybridization events. Using this method, we were able to observe selectivity of MoS2 for CGG repeats and distinguish nonpathogenic from disease-associated repeat lengths. The detection of CGG repeat sequences on inkjet printable 2D MoS2 surfaces is a forward step toward developing chip-based rapid and label-free sensors for the detection of repeat expansion sequences.

Systematic microsatellite repeat expansion cloning and validation.

Approximately 3% of the human genome is composed of short tandem repeat (STR) DNA sequence known as microsatellites, which can be found in both coding and non-coding regions. When associated with genic regions, expansion of microsatellite repeats beyond a critical threshold causes dozens of neurological repeat expansion disorders. To better understand the molecular pathology of repeat expansion disorders, precise cloning of microsatellite repeat sequence and expansion size is highly valuable. Unfortunately, cloning repeat expansions is often challenging and presents a significant bottleneck to practical investigation. Here, we describe a clear method for seamless and systematic cloning of practically any microsatellite repeat expansion. We use cloning and expansion of GGGGCC repeats, which are the leading genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), as an example. We employ a recursive directional ligation (RDL) technique to build multiple GGGGCC repeat-containing vectors. We describe methods to validate repeat expansion cloning, including diagnostic restriction digestion, PCR across the repeat, and next-generation long-read MinION nanopore sequencing. Validated cloning of microsatellite repeats beyond the critical expansion threshold can facilitate step-by-step characterization of disease mechanisms at the cellular and molecular level.