Molecular Biology

Plant genome editing is a powerful approach for modifying plant DNA to investigate gene function and to engineer desirable traits. Several genome-editing technologies have been developed, among which CRISPR/Cas systems and transcription activator-like effector nucleases (TALENs) are widely used to introduce targeted double-stranded DNA breaks. While CRISPR/Cas systems are highly efficient for nuclear genome editing, their application to plant organellar genomes remains limited, largely due to difficulties in guide RNA delivery into mitochondria and chloroplasts. Here, we present a detailed and reproducible protocol for constructing TALEN-based binary vectors for targeted genome editing in Arabidopsis thaliana. This protocol describes the assembly of TALE repeat arrays, the generation of nuclear-, mitochondrial-, and plastid-targeted TALEN expression vectors using MultiSite Gateway cloning, and subsequent Agrobacterium-mediated plant transformation and genotyping. The workflow enables the production of nTALENs, mitoTALENs, and ptpTALENs using a unified vector design strategy. In addition, the protocol briefly outlines the construction principles of TALE-based cytidine deaminases (TALECDs) for targeted C-to-T base editing in plant organellar genomes. The protocol provides a flexible and robust framework for plant nuclear and organellar genome editing and can be readily adapted to different target genes and experimental purposes. Its modular design and compatibility with standard molecular cloning techniques make it accessible to laboratories aiming to perform precise genome manipulation in plants.

Nanobodies are recombinant single-domain antibodies (VHHs) derived from the heavy chain–only subset of camelid immunoglobulins that can be reverse-engineered into bivalent antibodies by fusion to immunoglobulin Fc constant regions. Mammalian cells are the system of choice to produce VHH-Fcs to ensure authentic folding and post-translation glycosylation of the expressed VHH-Fcs. In a recent project to find neutralising VHH-Fc binders to the spike proteins of SARS-CoV-2 viruses, we identified a need for rapid expression and purification of multiple VHH-Fc fusions from nanobodies selected by phage display. Here, we present a protocol for the construction of expression vectors by parallel ligase-independent cloning, transient small-scale expression in mammalian cells (4 mL culture volume), screening antigen-binding activity, and midi-scale purification (30 mL culture volume) for downstream activity assays. The workflow is completely transferable between different vector formats, of which three are described herein: Fc fusion dimers, monomeric CD4 fusions, and His-tagged monomers.

We established a step-by-step approach for generating a single-nucleotide mutation in the promoter region of an immune regulatory gene in human monocyte THP-1 cells by employing a plasmid-based CRISPR-Cas9 system delivered via transfection with a homology-directed repair template DNA (HDR). Key steps include designing a single-guide RNA (sgRNA), cloning it into a CRISPR plasmid encoding the Cas9 protein, transfection of the plasmid constructs along with single-stranded oligonucleotide repair template (ssODNs) into THP-1 cells, followed by selection and validation. This approach provides a precise and relevant model to investigate the role of single polymorphisms in the regulation of inflammatory gene expression in human monocytes. In addition to the rs1024611 single-nucleotide polymorphism (SNP), this CRISPR/Cas9-based strategy is broadly applicable to functional studies of noncoding and coding variants in innate immune genes.

Engineering of microbial cells, including E. coli, is essential in prototyping genetic designs used in numerous applications throughout synthetic biology. While many advanced genome editing tools, such as CRISPR-based tools, offer new capabilities with genetically recalcitrant organisms, these tools often do not offer an immediate advantage in readily manipulated microbes, such as E. coli, especially when scarless modifications are not critical. We describe a comprehensive recombineering tutorial that we commonly use for multiplex engineering of E. coli using antibiotic markers. We leverage a group of 15 antibiotic resistance cassettes, most of which can be readily included when designing double-stranded DNA donors intended for recombineering and purchased from several vendors. Using these methods, 10–15 defined modifications to a single host strain can be achieved in less than three weeks, using two-day editing cycles. We discuss sequences and protocols as well as the optimal design of genetic modifications and the associated DNA.

In wheat and other crops, some genes display presence/absence variation, and it is occasionally beneficial to select for the absent allele to remove a functional gene. However, current high-throughput genotyping methods used to detect the absence of genes tend to be inconsistent and inconclusive. Kompetitive allele-specific PCR (KASP) and PCR allele competitive extension (PACE) are two well-established methods for allele-specific polymerase chain reaction (AS-PCR) assays, each using fluorescence resonance energy transfer (FRET) to generate a signal for each allele, typically targeting biallelic single-nucleotide polymorphisms. KASP and PACE methods are more difficult to apply to alleles with presence/absence variation because the lack of amplification of the absent allele is indistinguishable from a failed PCR. Here, we present a multiplex fluorescence-based absent allele–specific amplification (FAASA) method using the PACE marker system (compatible with KASP markers) to detect the absence of one particular or all alleles of a target sequence using a primer mix consisting of one target-specific primer pair (TSP) and a second primer set specific to a highly conserved endogenous gene known as a core gene–specific primer pair (CGSP). The forward primer of each pair is tagged with a 5′ terminal tail complementary to dye-labeled oligonucleotides in commercially available FRET cassettes. Lines that amplify only the core gene do not carry the target, while lines that amplify both the core gene and the target carry alleles of both the core gene and the target. The inclusion of the CGSPs allows researchers to confidently distinguish lines with absent alleles of the target from lines with failed PCR reactions, which can happen due to various reasons, including inadequate DNA quality or quantity.

Agrobacterium rhizogenes–mediated hairy root transformation provides a rapid platform for gene function analysis prior to stable whole-plant transformation. However, most existing hairy root transformation methods rely on tissue culture and require chemical or fluorescence-based selection, which increases experimental complexity. Here, we describe a tissue culture–free soybean hairy root transformation protocol incorporating the RUBY visual reporter system. While this work does not introduce a new transformation concept, it presents a streamlined implementation of established soybean hairy root methodologies that emphasizes procedural simplicity, reduced handling, and faster access to functional root material. Transgenic roots expressing RUBY can be directly identified by red pigmentation with the naked eye. In RUBY-positive roots, candidate genes driven by the CaMV 35S promoter showed higher expression levels than those in empty-vector controls, indicating that the system supports effective gene expression. Using this procedure, clearly identifiable transgenic hairy roots can be obtained within 20 days. Overall, this protocol simplifies induction and screening while reducing operational complexity and equipment requirements.

Extrachromosomal circular DNA (eccDNA) is a type of circular DNA that exists independently of chromosomes and has garnered significant attention in various fields, particularly in the context of smaller eccDNAs, which have considerable roles in gene regulation through various mechanisms. Current methods such as Circle-Seq and 3SEP can enrich small eccDNAs during sample preparation, but most bioinformatics pipelines remain challenging, exhibiting low accuracy and efficiency. This protocol describes the detailed workflow of a newly developed bioinformatics analysis pipeline, named EccDNA Caller based on Consecutive Full Pass (ECCFP), to accurately identify eccDNA from long-read Nanopore sequencing data. Compared to other pipelines, ECCFP significantly improves detection sensitivity, accuracy, and runtime efficiency. The process includes raw data quality control, trimming of adapters and barcodes, alignment to a reference genome, and identification of eccDNA, with detailed results encompassing accurate positioning of eccDNA, consensus sequences, and variants of individual eccDNA.

Transcription factors (TFs) regulate gene expression by binding to cis-regulatory elements in the genome. Understanding transcriptional regulation requires genome-wide characterization of TF occupancy across different chromatin contexts, yet simultaneous assessment of TF binding for multiple factors remains technically challenging. Here, we describe a detailed and reproducible protocol for cFOOT-seq, a cytosine deaminase–based genomic footprinting assay by sequencing, which enables antibody-independent, base-resolution profiling of chromatin accessibility, nucleosome organization, and TF occupancy. In cFOOT-seq, the double-stranded DNA (dsDNA) cytosine deaminase SsdA_tox converts cytosine to uracil in accessible chromatin, whereas TF binding and nucleosome occupancy locally protect DNA from deamination. Using the FootTrack analysis framework, deamination patterns generated by cFOOT-seq are quantitatively analyzed to derive standardized footprint and chromatin organization profiles at base resolution across the genome. Because cFOOT-seq preserves genomic DNA integrity during deamination-based footprinting, it is compatible with ATAC-seq-based chromatin enrichment. ATAC-combined implementations reduce sequencing depth requirements and improve scalability for footprint-focused analyses, supporting applications in low-input and single-cell settings. This protocol provides a practical framework for genome-wide TF footprint profiling and can be readily applied to dissect gene regulatory mechanisms in development, immunity, and disease, including cancer.

Amphibian retinas contain “green” rods, which are rod-shaped photoreceptors with a cone-type visual pigment. These rods are considered a potentially transitional photoreceptor type, but their phototransduction cascade’s molecular composition has remained uncertain. Here, we present a streamlined electrophysiology-molecular workflow that enables the rapid spectral identification, physical capture, and targeted single-cell reverse transcription-polymerase chain reaction (RT-PCR) of individual amphibian photoreceptors. After suction-pipette spectral screening under alternating red and green illumination, electrophysiologically identified cells are isolated and processed directly for reverse transcription and PCR. Coupling real-time functional phenotyping with sensitive molecular profiling provides a practical tool for resolving photoreceptor molecular heterogeneity and investigating evolutionary transitions between rod and cone phenotypes.

DNA epigenetic modifications play crucial roles in regulating gene expression and cellular function across diverse organisms. Among them, 5-glyceryl-methylcytosine (5gmC), a unique DNA modification first discovered in Chlamydomonas reinhardtii, represents a novel link between redox metabolism and epigenetic regulation. Accurate genome-wide detection of 5gmC is essential for investigating its biological functions, yet no streamlined method has been available. Here, we present deaminase-assisted sequencing (DEA-seq), a simple and robust approach for base-resolution mapping of 5gmC. DEA-seq employs a single DNA deaminase that efficiently converts unmodified cytosines (C) and 5-methylcytosine (5mC) into uracils or thymines, while leaving 5gmC intact. This selective resistance generates a clear sequence signature that enables precise identification of 5gmC sites across the genome. The method operates under mild reaction conditions and is compatible with low-input DNA, minimizing sample loss and improving detection sensitivity. Overall, DEA-seq provides an accessible, efficient, and highly accurate protocol for profiling 5gmC, offering clear advantages in workflow simplicity, DNA integrity, and analytical performance.