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BioTechniquesVol. 58, No. 4 BioSpotlight / CitationsOpen AccessBioSpotlight / CitationsNathan S. Blow & Nijsje DormanNathan S. BlowSearch for more papers by this author & Nijsje DormanSearch for more papers by this authorPublished Online:3 Apr 2018https://doi.org/10.2144/000114271AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail Crispr cloningCloning via restriction enzymes or PCR is a mainstay technique in molecular biology labs around the globe. The challenge when working with restriction enzymes is that they are specific, cleaving in only certain locations, while PCR approaches can lead to unintended sequence changes and are limited by possible amplicon size. In this issue, Wang et al. demonstrate for the first time that direct, seamless cloning of DNA fragments into any sequence location can be achieved through the use of CRISPR/Cas9 nuclease cleavage along with subsequent Gibson assembly. The authors demonstrate their combination technique by linearizing a 22 kb plasmid with the Cas9 enzyme and a specific single guide RNA (sgRNA) in vitro and then inserting a DNA fragment using Gibson assembly. This new technique opens doors for researchers interested in working with larger vectors such as cosmids, adenoviral vectors, and bacterial artificial chromosome (BAC) plasmids, or modifying existing vector constructs when the existing methods are not suitable or sufficient.See “CRISPR/Cas9 nuclease cleavage combined with Gibson assembly for seamless cloning”Reagents to explore genome architectureIt's become clear in recent years that the three-dimensional organization of genomic DNA is critical when it comes to determining gene expression patterns and genome function. Much of this knowledge has been gained through the use of chromosome conformation capture (3C, 4C, 5C, Hi-C, etc.) techniques that measure long-range interactions between different regions of the genome. While these techniques have provided unique insights into genome regulation, there has yet to be a systematic evaluation and optimization of the two critical enzymes used in chromosome conformation capture assays: T4 DNA ligase and DNA polymerase. In this issue, Schwartz et al. perform a comparison of different commercially available ligases and polymerases. The authors tested 4 different T4 DNA ligases and 13 different DNA polymerases, identifying the most efficient and specific combinations of each and thereby providing researchers lists of well-validated reagents, which should facilitate more robust, reproducible, and less costly chromosome conformation capture analysis in the future.See “Comparative analysis of ligases and polymerases used in chromosome conformation capture assays”Cell-permeable probe to fluorescently label his-tagsThough best known for affinity purification, the His-tag can also be conjugated with small-molecule fluorescent labels. However, no fluorophore has been shown capable of rapid intracellular labeling of His-tagged proteins. Lai et al. change all that with the Ni-NTA-AC probe, comprising a nitrilotriacetate moiety conjugated with a coumarin fluorophore and an attached arylazide. Although binding is relatively weak, UV irradiation photoactivates the arylazide, which covalently binds the protein and enhances fluorescence 13-fold. In tests in HeLa cells with a His-tagged cytosolic protein, adding Ni-NTA-AC to the medium led to intracellular blue fluorescence within 2 minutes, indicating rapid transmembrane movement and protein labeling. No toxicity from the fluorophore or UV irradiation was observed. Furthermore, in studies with a 15-kDa DNA binding domain, the method gave the expected nuclear localization, unlike labeling via an RFP fusion. The new label also works in plant tissue, and other colors of His-tag–binding fluorophore are in development.Y.T. Lai et al. Rapid labeling of intracellular His-tagged proteins in living cells. Proc Natl Acad Sci U S A. [Epub ahead of print, Feb 23, 2015; doi: 10.1073/pnas.1419598112]Whole-mount single-molecule fish in a vertebrate embryoAnalyzing gene expression at the single-cell level reveals stochastic variations thought to underlie adaptability. Quantitative RT-PCR and RNA-Seq measure these fluctuations, but lack the spatial resolution afforded by single-molecule FISH (smFISH). Although smFISH has been performed in tissue from vertebrates, to date whole-mount smFISH protocols are limited to invertebrates. To fill this gap, Oka and Sato took elements of smFISH methods for cultured cells and Drosophila embryos and combined them with features of traditional in situ hybridization in whole mounts of zebrafish embryos. By optimizing pretreatment, fixation, and washes, the authors could detect transcripts just 720 nucleotides long (close to the theoretical minimum length of 700 nucleotides in this system) and genes that are expressed at fewer than 50 copies per cell. Experiments in which in vitro–transcribed RNA was injected and then probed gave confidence that each smFISH dot corresponds to one transcript. The authors expect their method will also be applicable to other vertebrate model systems.Y. Oka and T.N. Sato. 2015. Whole-mount single molecule FISH method for zebrafish embryo. Sci Rep. 5:8571.FiguresReferencesRelatedDetails Vol. 58, No. 4 Follow us on social media for the latest updates Metrics History Published online 3 April 2018 Published in print April 2015 Information© 2015 Author(s)PDF download