Molecular Biology


    Protocols in Current Issue
    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: 756, 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: 683, 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: 1095, 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: 1972, 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: 2660, Q&A: 0
    [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: 2154, 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 ...
    Improving CRISPR Gene Editing Efficiency by Proximal dCas9 Targeting
    Authors:  Fuqiang Chen, Xiao Ding, Yongmei Feng, Timothy Seebeck, Yanfang Jiang and Gregory D Davis, date: 08/05/2017, view: 2463, Q&A: 0
    [Abstract] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems function as an adaptive immune system in bacteria and archaea for defense against invading viruses and plasmids (Barrangou and Marraffini, 2014). The effector nucleases from some class 2 CRISPR-Cas systems have been repurposed for heterologous ...
    Endogenous C-terminal Tagging by CRISPR/Cas9 in Trypanosoma cruzi
    Authors:  Noelia Lander, Miguel A. Chiurillo, Aníbal E. Vercesi and Roberto Docampo, date: 05/20/2017, view: 4129, Q&A: 0
    [Abstract] To achieve the C-terminal tagging of endogenous proteins in T. cruzi we use the Cas9/pTREX-n vector (Lander et al., 2015) to insert a specific tag sequence (3xHA or 3xc-Myc) at the 3’ end of a specific gene of interest (GOI). Chimeric sgRNA targeting the 3’ end of the GOI is PCR-amplified and cloned into Cas9/pTREX-n vector. Then ...
    Multiplexed GuideRNA-expression to Efficiently Mutagenize Multiple Loci in Arabidopsis by CRISPR-Cas9
    Authors:  Julia Schumacher, Kerstin Kaufmann and Wenhao Yan, date: 03/05/2017, view: 3618, Q&A: 0
    [Abstract] Since the discovery of the CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein (Cas) as an efficient tool for genome editing in plants (Li et al., 2013; Shan et al., 2013; Nekrasov et al., 2013), a large variety of applications, such as gene knock-out, knock-in or transcriptional ...
    Identification of Methylated Deoxyadenosines in Genomic DNA by dA6m DNA Immunoprecipitation
    [Abstract] dA6m DNA immunoprecipitation followed by deep sequencing (DIP-Seq) is a key tool in identifying and studying the genome-wide distribution of N6-methyldeoxyadenosine (dA6m). The precise function of this novel DNA modification remains to be fully elucidated, but it is known to be absent from transcriptional start ...