Molecular Biology

CRISPR-Cas9 technology has become an essential tool for plant genome editing. Recent advancements have significantly improved the ability to target multiple genes simultaneously within the same genetic background through various strategies. Additionally, there has been significant progress in developing methods for inducible or tissue-specific editing. These advancements offer numerous possibilities for tailored genome modifications. Building upon existing research, we have developed an optimized and modular strategy allowing the targeting of several genes simultaneously in combination with the synchronized expression of the Cas9 endonuclease in the egg cell. This system allows significant editing efficiency while avoiding mosaicism. In addition, the versatile system we propose allows adaptation to inducible and/or tissue-specific edition according to the promoter chosen to drive the expression of the Cas9 gene. Here, we describe a step-by-step protocol for generating the binary vector necessary for establishing Arabidopsis edited lines using a versatile cloning strategy that combines Gateway^® and Golden Gate technologies. We describe a versatile system that allows the cloning of as many guides as needed to target DNA, which can be multiplexed into a polycistronic gene and combined in the same construct with sequences for the expression of the Cas9 endonuclease. The expression of Cas9 is controlled by selecting from among a collection of promoters, including constitutive, inducible, ubiquitous, or tissue-specific promoters. Only one vector containing the polycistronic gene (tRNA-sgRNA) needs to be constructed. For that, sgRNA (composed of protospacers chosen to target the gene of interest and sgRNA scaffold) is cloned in tandem with the pre-tRNA sequence. Then, a single recombination reaction is required to assemble the promoter, the zCas9 coding sequence, and the tRNA-gRNA polycistronic gene. Each element is cloned in an entry vector and finally assembled according to the Multisite Gateway^® Technology. Here, we detail the process to express zCas9 under the control of egg cell promoter fused to enhancer sequence (EC1.2en-EC1.1p) and to simultaneously target two multiple C2 domains and transmembrane region protein genes (MCTP3 and MCTP4, respectively at3g57880 and at1g51570), using one or two sgRNA per gene.

Precision-cut lung slices (PCLS), ex vivo 3D lung tissue models, have been widely used for various applications in lung research. PCLS serve as an excellent intermediary between in vitro and in vivo models because they retain all resident cell types within their natural niche while preserving the extracellular matrix environment. This protocol describes the TReATS (TAT-Cre recombinase-mediated floxed allele modification in tissue slices) method that enables rapid and efficient gene modification in PCLS derived from adult floxed animals. Here, we present detailed protocols for the TReATS method, consisting of two simple steps: PCLS generation and incubation in a TAT-Cre recombinase solution. Subsequent validation of gene modification involves live staining and imaging of PCLS, quantitative real-time PCR, and cell viability assessment. This four-day protocol eliminates the need for complex Cre-breeding, circumvents issues with premature lethality related to gene mutation, and significantly reduces the use of animals. The TReATS method offers a simple and reproducible solution for gene modification in complex ex vivo tissue-based models, accelerating the study of gene function, disease mechanisms, and the discovery of drug targets.

Study of gene function in eukaryotes frequently requires data on the impact of the gene when it is expressed as a transgene, such as in ectopic or overexpression studies. Currently, the use of transgenic constructs designed to achieve these aims is often hampered by the difficulty in distinguishing between the expression levels of the endogenous gene and its transgene equivalent, which may involve either laborious microdissection to isolate specific cell types or harvesting tissue at narrow timepoints. To address this challenge, we have exploited a feature of the Golden Gate cloning method to develop a simple, restriction digest–based protocol to differentiate between expression levels of transgenic and endogenous gene copies. This method is straightforward to implement when the endogenous gene contains a Bpi1 restriction site but, importantly, can be adapted for most genes and most other cloning strategies.

Key features

• This protocol was developed to determine the expression level of an ectopically expressed transcription factor with broad native expression in all surrounding tissues.

• The method described is most directly compatible with Golden Gate cloning but is, in principle, compatible with any cloning method.

• The protocol has been developed and validated in the model plant Arabidopsis thaliana but is applicable to most eukaryotes.

Graphical overview

To optimize differentiation protocols for stem cell-based in vitro modeling applications, it is essential to assess the change in gene expression during the differentiation process. This allows controlling its differentiation efficiency into the target cell types. While RNA transcriptomics provides detail at a larger scale, timing and cost are prohibitive to include such analyses in the optimization process. In contrast, expression analysis of individual genes is cumbersome and lengthy.

Here, we developed a versatile and cost-efficient SYBR Green array of 27 markers along with two housekeeping genes to quickly screen for differentiation efficiency of human induced pluripotent stem cells (iPSCs) into excitatory cortical neurons. We first identified relevant pluripotency, neuroprogenitor, and neuronal markers for the array by literature search, and designed primers with a product size of 80-120 bp length, an annealing temperature of 60°C, and minimal predicted secondary structures. We spotted combined forward and reverse primers on 96-well plates and dried them out overnight. These plates can be prepared in advance in batches and stored at room temperature until use. Next, we added the SYBR Green master mix and complementary DNA (cDNA) to the plate in triplicates, ran quantitative PCR (qPCR) on a Quantstudio 6 Flex, and analyzed results with QuantStudio software.

We compared the expression of genes for pluripotency, neuroprogenitor cells, cortical neurons, and synaptic markers in a 96-well format at four different time points during the cortical differentiation. We found a sharp reduction of pluripotency genes within the first three days of pre-differentiation and a steady increase of neuronal markers and synaptic markers over time. In summary, we built a gene expression array that is customizable, fast, medium-throughput, and cost-efficient, ideally suited for optimization of differentiation protocols for stem cell-based in vitro modeling.

Cells are the complex product of gene expression programs that involve the coordinated transcription of thousands of genes controlled by cis-regulatory elements (cis-REs). Therefore, identification of cis-REs is the key to decipher the mechanisms underlying the regulation of gene expression. Here, we describe a simple and time-effective protocol of fine-mapping cis-REs by using an electrophoresis mobility shift assay (EMSA)-based, in vitro, high-throughput (HTP) technique called regulatory element-sequencing (Reel-seq). Reel-seq can be applied to identify cis-REs at a high resolution and sensitivity over large genome regions, in a systematic and continuous manner. It can be used to prioritize candidate cis-REs as a complement to the existing approaches, such as massive parallel reporter assay (MPRA), chromatin immunoprecipitation DNA-sequencing (ChIP-seq), and the assay for transposase-accessible chromatin sequencing (ATAC-seq).

Graphical abstract:

Generation of the Reel-seq Library 1 and 2 (A) and identification of cis-REs by an electrophoresis mobility shift assay (EMSA)-based Reel-seq screen (B). NE: nuclear extract; NGS: next generation sequencing.

The ability to identify the role of a particular gene within a system is dependent on control of the expression of that gene. In this protocol, we describe a method for stable, conditional expression of Nod-Like receptors (NLRs) in THP-1 cells using a lentiviral expression system. This system combines all the necessary components for tetracycline-inducible gene expression in a single lentivector with constitutive co-expression of a selection marker, which is an efficient means for controlling gene expression using a single viral infection of cells. This is done in a third generation lentiviral expression platform that improves the safety of lentiviruses and allows for greater gene expression than previous lentiviral platforms. The lentiviral expression plasmid is first engineered to contain the gene of interest driven by a TRE (tetracycline response element) promoter in a simple gateway cloning step and is then co-transfected into HEK293T cells, along with packaging and envelope plasmids to generate the virus. The virus is used to infect a cell type of interest at a low MOI so that the majority of the transduced cells contain a single viral integration. Infected cells are grown under selection, and viral integration is validated by qPCR. Gene expression in stably transduced cells is induced with doxycycline and validated by qPCR, immunoblot, and flow cytometry. This flexible lentiviral expression platform may be used for stable and robust induction of a gene of interest in a range of cells for multiple applications.

Graphic abstract:

Schematic overview of lentiviral transduction of THP-1 cells.

The abilities to mark and manipulate specific cell types are essential for an increasing number of functional, structural, molecular, and developmental analyses in model organisms. In a few species, this can be accomplished by germline transgenesis; in other species, other methods are needed to selectively label somatic cells based on the genes that they express. Here, we describe a method for CRISPR-based somatic integration of reporters or Cre recombinase into specific genes in the chick genome, followed by visualization of cells in the retina and midbrain. Loci are chosen based on an RNA-seq-based cell atlas. Reporters can be soluble to visualize the morphology of individual cells or appended to the encoded protein to assess subcellular localization. We call the method eCHIKIN for electroporation- and CRISPR-mediated Homology-instructed Knock-IN.

Nowadays, CRISPR (clustered regularly interspaced short palindromic repeats) and the CRISPR-associated protein (Cas9) system play a major role in genome editing. To target the desired sequence of the genome successfully, guide RNA (gRNA) is indispensable for the CRISPR/Cas9 system. To express gRNA, a plasmid expressing the gRNA sequence is typically constructed; however, construction of plasmids involves much time and labor. In this study, we propose a novel procedure to express gRNA via a much simpler method that we call gRNA-TES (gRNA-transient expression system). This method employs only PCR, and all the steps including PCR and yeast transformation can be completed within 1 day. In comparison with the plasmid-based gRNA delivery system, the performance of gRNA-TES is more effective, and its total time and cost are significantly reduced.

Investigation of bacterial gene regulation upon environmental changes is still a challenging task. For example, Vibrio cholerae, a pathogen of the human gastrointestinal tract, faces diverse transient conditions in different compartments upon oral ingestion. Genetic reporter systems have been demonstrated to be extremely powerful tools to unravel gene regulation events in complex conditions, but so far focused mainly on gene induction. Herein, we describe the TetR-controlled recombination-based in vivo expression technology TRIVET, which allows detection of gene silencing events. TRIVET resembles a modified variant of the in vivo expression technology (IVET) as well as recombination-based in vivo expression technology (RIVET), which were used to identify conditional gene induction in several bacteria during host colonization. Like its predecessors, TRIVET is a single cell based reporter system, which allows the analysis of bacterial gene repression in a spatiotemporal manner via phenotypical changes in the resistance profile. Briefly, a promoterless tetR (encoding the transcriptional repressor TetR) can be integrated randomly into the bacterial genome via transposon mutagenesis or site-specific downstream of a promoter of interest via homologous recombination. Reduction of transcriptional expression of TetR results in a de-repression of the TetR-controlled resolvase TnpR, which in turn leads to excision of an antibiotic resistance cassette (also known as res-cassette) and altered resistance profile observable via streaking on ampicillin and kanamycin plates. This alteration can then be quantified as the ratio between resistant and non-resistant isolates. Furthermore, the newly introduced second reporter gene, a promoterless phoA (encoding the alkaline phosphatase PhoA) offers an additional validation step of the results via an independent colorimetric assay to measure enzyme activity. The protocol presented herein also offers an approach to identify the gene locus in case of the random screen for gene repression as well as a quantification of the conditional repression of a gene of interest. Although the current protocol is established for gene repression during host colonization, it can likely be adapted to study gene silencing under various conditions faced by a bacterium.

A viral vector that can safely and efficiently deliver large and diverse molecular cargos into cells is the holy grail of curing many human diseases. Adeno-associated virus (AAV) has been extensively used but has a very small capacity. The prokaryotic virus T4 has a large capacity but lacks natural mechanisms to enter mammalian cells. Here, we created a hybrid vector by combining T4 and AAV into one nanoparticle that possesses the advantages of both. The small 25 nm AAV particles are attached to the large 120 nm x 86 nm T4 head through avidin-biotin cross-bridges using the phage decoration proteins Soc (small outer capsid protein) and Hoc (highly antigenic outer capsid protein). AAV thus “piggy-backed” on T4 capsid, by virtue of its natural ability to enter many types of human cells efficiently acts as a “driver” to deliver large cargos associated with the T4 head. This unique T4-AAV hybrid vector approach could pave the way for the development of novel therapeutics in the future.