Molecular Biology

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    Protocols in Current Issue
    Flip-flop Mediated Conditional Gene Inactivation in Drosophila
    Authors:  Sathiya N. Manivannan, Priyanka Pandey and Sonal Nagarkar-Jaiswal, date: 02/05/2019, view: 842, Q&A: 0
    [Abstract] Mosaic analysis in Drosophila, an important tool to assess cellular phenotypes of mutants in an otherwise heterozygous background, relies on mitosis. Hence, it cannot be used to inactivate gene function in mitotically inactive, terminally differentiated cells such as neurons. To address this issue, we developed “Flip-flop”, a novel, ...
    flySAM Transgenic CRISPRa System Manual
    Authors:  Yu Jia, Da Shen, Xia Wang, Jin Sun, Ping Peng, Rong-Gang Xu, Bowen Xu and Jian-Quan Ni , date: 01/20/2019, view: 1222, Q&A: 0
    [Abstract] Powerful and general methods that can enhance gene expression are useful to systematically study gene function. To date, compared with the methods in generating loss-of-function mutants, methods to achieve gain-of-function are limited. The entire field in Drosophila has relied heavily on the Gal4/UAS:cDNA overexpression system developed ...
    CRISPR-Cas9 Mediated Genome Editing in Drosophila
    Authors:  Ping Peng, Xia Wang, Da Shen, Jin Sun, Yu Jia, Rong-Gang Xu, Li-Fei Zhu and Jian-Quan Ni , date: 01/20/2019, view: 1240, Q&A: 0
    [Abstract] In recent years, great progress has been made in the research of genome editing systems, one of which is the CRISPR-Cas9 system, a powerful technology that is applied to edit animal genome. Here, we describe a CRISPR-Cas9 mediated mutation protocol for efficiently and specifically editing genes in Drosophila. In this optimized system, the ...
    Single and Multiplexed Gene Editing in Ustilago maydis Using CRISPR-Cas9
    [Abstract] The smut fungus Ustilago maydis is an established model organism for elucidating how biotrophic pathogens colonize plants and how gene families contribute to virulence. Here we describe a step by step protocol for the generation of CRISPR plasmids for single and multiplexed gene editing in U. maydis. Furthermore, we describe the ...
    Design of Hybrid RNA Polymerase III Promoters for Efficient CRISPR-Cas9 Function
    Authors:  Joshua Misa, Cory Schwartz and Ian Wheeldon, date: 03/20/2018, view: 2805, Q&A: 0
    [Abstract] The discovery of the CRISPR-Cas9 system from Streptococcus pyogenes has allowed the development of genome engineering tools in a variety of organisms. A frequent limitation in CRISPR-Cas9 function is adequate expression levels of sgRNA. This protocol provides a strategy to construct hybrid RNA polymerase III (Pol III) promoters that ...
    Coupling Exonuclease Digestion with Selective Chemical Labeling for Base-resolution Mapping of 5-Hydroxymethylcytosine in Genomic DNA
    Authors:  Aurélien A. Sérandour, Stéphane Avner and Gilles Salbert, date: 03/05/2018, view: 2351, Q&A: 0
    [Abstract] This protocol is designed to obtain base-resolution information on the level of 5-hydroxymethylcytosine (5hmC) in CpGs without the need for bisulfite modification. It relies on (i) the capture of hydroxymethylated sequences by a procedure known as ‘selective chemical labeling’ (see Szulwach et al., 2012) and (ii) the digestion of the ...
    Genotyping-free Selection of Double Allelic Gene Edited Medaka Using Two Different Fluorescent Proteins
    Authors:  Yu Murakami, Satoshi Ansai, Akari Yonemura and Masato Kinoshita, date: 12/20/2017, view: 2693, Q&A: 0
    [Abstract] This protocol describes a simple genotyping using two different colors of fluorescent protein genes inserted at the target locus. This method makes it possible to determine the genotype of each individual simply by observing the fluorescence later than F1 generation.
    Genome Editing in Diatoms Using CRISPR-Cas to Induce Precise Bi-allelic Deletions
    Authors:  Amanda Hopes, Vladimir Nekrasov, Nigel Belshaw, Irina Grouneva, Sophien Kamoun and Thomas Mock, date: 12/05/2017, view: 4763, Q&A: 1
    [Abstract] Genome editing in diatoms has recently been established for the model species Phaeodactylum tricornutum and Thalassiosira pseudonana. The present protocol, although developed for T. pseudonana, can be modified to edit any diatom genome as we utilize the flexible, modular Golden Gate cloning system. The main steps include ...
    Cytosolic and Nuclear Delivery of CRISPR/Cas9-ribonucleoprotein for Gene Editing Using Arginine Functionalized Gold Nanoparticles
    Authors:  Rubul Mout and Vincent M. Rotello, date: 10/20/2017, view: 5412, Q&A: 1
    [Abstract] In this protocol, engineered Cas9-ribonucleoprotein (Cas9 protein and sgRNA, together called Cas9-RNP) and gold nanoparticles are used to make nanoassemblies that are employed to deliver Cas9-RNP into cell cytoplasm and nucleus. Cas9 protein is engineered with an N-terminus glutamic acid tag (E-tag or En, where n = the number of glutamic acid in ...
    Protocol for Construction of a Tunable CRISPR Interference (tCRISPRi) Strain for Escherichia coli
    Authors:  Xin-tian Li, Cindy Sou and Suckjoon Jun, date: 10/05/2017, view: 4264, Q&A: 0
    [Abstract] We present a protocol for construction of tunable CRISPR interference (tCRISPRi) strains for Escherichia coli. The tCRISPRi system alleviates most of the known problems of plasmid-based expression methods, and can be immediately used to construct libraries of sgRNAs that can complement the Keio collection by targeting both essential and ...



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