...about the role of methylation (Hrit et al. 2018, Kaas et al. 2013). Figure 2: CRISPR TET-based...CRISPR-based approach for targeted DNA demethylation. Cell Discov 2, 16009. https://doi.org/10.1038/celldisc...cause demethylation?  Dividing cells depend on DNA methyltransferase 1 (DNMT1) to copy methylation marks... proteins are not demethylases; they are dioxygenases that oxidize 5-methylcytosine to create rare DNA...complete DNA demethylation can be achieved by dCas9 alone, without TET (or any demethylase) fusion. This...site. So, when DNMT1 methylates the genome during DNA replication, it doesn’t methylate sites blocked by ...the original methylated strand is diluted to undetectable amounts: ergo, DNA demethylation! With optimization...

...as depicted in Lane 2. Controlling methylation Finally, you can control methylation by altering your choice... Dcm methylase adds a methyl group to the second cytosine of CCWGG EcoKI methylase adds a methyl group...contain 3 methylases that recognize and methylate different stretches of DNA: Dam methylase adds a methyl...information. Enzyme Dam methylation Dcm methylation EcoKI methylation ApaI Not affected Blocked ...questions is the same--methylation! Read on to learn about how DNA methylation may affect your restriction...The other component of these systems, methyltransferases, methylate DNA at particular sequences to prevent...restriction modification systems containing methyltransferases that add methyl groups to specific DNA sequences ...

...SARS-CoV-2 spike pseudotyping, visit the SARS-CoV-2 Pseudotyped Virus collection page. SARS-CoV-2 detection...of SARS-CoV-2 entry and become upregulated during viral infection.  Expression of SARS-CoV-2 viral proteins...labs in over 75 countries. Figure 1: SARS-CoV-2 proteins and other components. Image from Maya Kostman... protein, variants, and pseudotyping The SARS-CoV-2 virus depends on its spike protein to bind ACE2, the...of infection and how to interfere with it in a BSL-2 lab instead of a BSL-3 lab. Jesse Bloom’s lab published...reagents for pseudotyping lentivirus with the SARS-CoV-2 spike protein. They deposited the plasmids used for...individuals are at neutralizing the different SARS-CoV-2 spike variants. You can learn more about their research...

...same site, is 2’-fluoro (2’-F), which substitutes a fluorine for the hydroxyl group at the 2’ position. ...most common of these is 2’-O-methylation, where a methyl group is added to the 2’ hydroxyl group of the...together, for example phosphorothioates with 2’-O-methyl modifications, proved more stability than just...generated by introduction of a single photocleavable 2-nitrobenzyl linker in the 20-bp targeting region of...linkages, unlocked nucleic acids, and a constrained ethyl.  Can’t decide which RNA backbone modification is...similar advantages, bridged nucleic acids with N-methyl substitutions are slightly more efficient and less...

...for the 2+ million DHS sites. See below for further details. DNA is a 3D Fractal Figure 2: DNA compaction...: 1. Find Enhancers that have NANOG binding sites 2. Design a CRISPR screen to target and disrupt the ...NANOG enhancers 3. CRISPR out the enhancers from step 2. See which ESCs die or differentiate Step 3 reveals...expression for NANOG enhancers, we’re ready for step 2: design a CRISPR screen to disrupt these enhancers...to CRISPR out the enhancers we identified in step 2. The result is a heterogenous population of stem cells... region that has mono-methylated and acetylated histones, but not tri-methylated histones, functions as...denote enhancers as follows: (mono_methylation + acetylation) - tri_methylation In the next section we’ll ...

.... Diagnostic restriction digests are comprised of 2 separate steps: 1) incubating your DNA with restriction...which cleave the DNA molecules at specific sites and 2) running the reaction on an agarose gel to determine...including a 1.2 kb insert. The plasmid was digested with 2 unique enzymes (HindIII and BamHI) and run on an agarose...to serve as a reference band. The uncut DNA (lane 2) shows 3 possible plasmid conformations, with relaxed...Watch out for methylation issues. Enzymes like XbaI and ClaI are sensitive to methylation and their activity...will need to purify your DNA from a dcm or dam methylation-deficient bacterial strain such as JM110 or INV110...

...//doi.org/10.1002/1096-8628(200022)97:2<147::aid-ajmg6>3.0.co;2-o Baker SA, Chen L, Wilkins AD, Yu P, ...Syndrome? Caused by a mutation in the methyl-CpG binding protein 2 (MECP2) gene, Rett Syndrome is a rare...mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat Genet 23:185–188 . https://doi.... RE, Zoghbi HY (2000) Rett syndrome: Methyl-CpG-binding protein 2 mutations and phenotype-genotype correlations...et al., 1999). We now know that MECP2 binds to methylated CpG dinucleotides and CAC trinucleotides during...

...of conventional gene targeting is lowered to about 2-3 months. But certain models remain more difficult...included in the final ssDNA template. Figure 2: Overview of ivTRT to generate an ssDNA repair template... floxed conditional allele requires you to target 2 loxP sites to intronic regions 0.5 - 0.8 kb apart....Typically, Easi-CRISPR works best with insertions of 2 kb or less. Luckily, most of the commonly used cassettes... induction systems (ttA or rtTA) are between 1 to 2 kb in length. There are plenty of Addgene plasmid ...create the ssDNA construct as described in Figure 2.  You can also use Addgene plasmids to purify Cas9...directed repair. Using long ssDNA donors, the Gurumurthy and Ohtsuka groups have obtained an average knock-in...

... to the lysosome where they are finally degraded (2, 3). Once you've created your fusion protein, it's...examples include the ImageJ plugins JACoP and Coloc 2, BioImageXD, and customized CellProfiler pipelines...colocalization analysis example is given in Figure 2 (adapted from 4) - Dunn et al. 2011 provides an in-depth...Since colocalization relies upon the detection of 2 independent fluorescent signals and their potential...here! References 1. Allen Institute Plasmid Pages 2. Cooper GM. 2000. The Cell: A Molecular Approach. ... in protein localization and transport between healthy and diseased states can also provide interesting... the function of your protein of interest in a healthy or diseased cell. We hope you find the resources...

...repository of tools for SAR-CoV-2 research. Two new antibodies, Anti-SARS-CoV-2 Nucleocapsid Protein [mBG17...Anti-SARS-CoV-2 Nucleocapsid Protein [mBG86], target distinct epitopes on the SARS-CoV-2 nucelocapsid ...Anti-SARS-CoV-2 Antibodies here! Terry, J.S., et al. (2021). Development of a SARS-CoV-2 nucleocapsid ...prime editor PE7 Antibodies to facilitate SARS-CoV-2 research New viral vector preps of Voltron2 voltage... incorporates La’s N-terminal domain, PE7 (Figure 2).  PE7 shows enhanced editing efficiencies in a number...editor. Find PE7 plasmids here!     Figure 2: PE7 improves prime editing. A) Schematic of PE7 prime...PMID: 38570691. Antibodies to facilitate SARS-CoV-2 research   By Ashley Waldron Ready-to-use recombinant...

...Citorik Plasmids here). In both cases, they used 1-2 CRISPR crRNAs targeting sequences found only in certain...resensitize antibiotic-resistant bacteria (Figure 2). The first phage introduced is lysogenic, integrating...machinery targeting 1) the given resistance gene and 2) a second lytic phage. Bacteria that receive this ...bacteria have previously flourished.   Figure 2: Two-phage strategies to resensitize bacteria to antibiotics...237163. Find plasmids from this paper at Addgene. 2. Bikard, David, et al. “Exploiting CRISPR-Cas nucleases...References 1. Citorik, Robert J., Mimee, Mark, and Timothy K. Lu. “Sequence-specific antimicrobials using ... Hiroki, Sebastien Lemire, Diana P. Pires, and Timothy K. Lu. “Engineering Modular Viral Scaffolds for...

...plasmid-based expression requires serial transfection of 1 or 2 plasmids that express the reprogramming factors of...for generating iPSCs by plasmid transfection where 2 of 11 clones tested had plasmid integration. Another...understanding the mechanisms of reprogramming, and 2) studies with clinical endpoints. In the first scenario...PubMed PMID: 27764162. PubMed Central PMCID: 5072705. 2. Sommer, Cesar A., and Gustavo Mostoslavsky. "The ...challenges." Journal of Cellular Physiology 228, no. 2 (2012): 267-75. PubMed PMID: 22767332.  3. Kim, Dohoon...PubMed PMID: 23546745. PubMed Central PMCID: 4176696. 2. Yusa, Kosuke, Roland Rad, Junji Takeda, and Allan...generation. Expert opinion on biological therapy, 10 2, 231-42. PubMed PMID: 20088717. PubMed Central PMCID...

...host range plasmids. FEMS Microbiology Letters, 348(2), 87–96. https://doi.org/10.1111/1574-6968.12241  ...mobilization of broad host-range IncQ plasmids. Plasmid, 62(2), 57–70. https://doi.org/10.1016/j.plasmid.2009.05.001...negative hosts. Biotechnology and Bioengineering, 106(2), 326–332. https://doi.org/10.1002/bit.22695  Schmidhauser...Legionella pneumophila, Methylophilus methyltrophus, Methylococcus methanolicus, Methylosinus trichosporium, Myxococcus...Klebsiella aerogenes, K. pneumoniae, Methylophilus methylotrophus, Moraxella spp., Mycobacterium aurum...Klebsiella spp., Legionella pneumophila, Methylophilus methylotrophus, Myxococcus virescens, M. xanthus, Proteus...eutrophus, Azotobacter vinelandii, Brevibacterium methylicum, Caulobacter crescentus, Desulfovibrio vulgaris...

...Table 2: Lab strains of E. coli Strain Natural resistance Primary Use Genotype ccdB Survival 2 T1R ...resistance to tetracycline Additionally, Table 2 provides a quick reference for some of the popular... mk+) Unmethylated DNA not degraded, cell still can methylate DNA   hsdS(rk-,mk-) Unmethylated DNA not...Consequence dam DNA adenine methylase mutation (GATC) Preparing unmethylated DNA, important when trying... XbaI) dcm DNA cytosine methylase mutation (CCWGG) Preparing unmethylated DNA, important when trying...pathway that cleaves methylated cytosine DNA Allows for uptake of foreign (methylated) DNA mrr, Δ(mcrC-mrr...that cleaves methylated adenine or cytosine DNA Allows for uptake of foreign (methylated) DNA recA, ...

...Figure 2: dCas9-Tet1 demethylates cytosines at promoters and enhancers. This targeted demethylation is associated...CRISPR-based approach for targeted DNA demethylation. Cell Discovery 2: . https://doi.org/10.1038/celldisc...pX330a dCas9-LSD1 for targeted removal of H3K4me1/2 and H3K9me2 histone modifications. Like the TALE-LSD1...fused a TALEN to the Tet1 demethylase catalytic domain, could mediate demethylation and induce transcription... fused a TAL effector to LSD1 histone demethylase to demethylate enhancer regions (Mendenhall et al., ...Tet1 demethylase Ronggui Hu’s lab has created pdCas9-Tet1-CD for targeted cytosine demethylation in mammalian... dCas9-DNMT3A methylates cytosines at promoters and enhancers. This targeted methylation is associated...

... the two DNA strands and releasing topoisomerase (2).  These days commercially available TOPO kits provide...Taq polymerase using your favorite PCR protocol.  2. Set Up TOPO Cloning Reaction: Mix together the PCR...PubMed PMID: 1658796. PubMed Central PMCID: PMC52876. 2. Novel approach to molecular cloning and polynucleotide...ability of complementary basepairs adenine (A) and thymine (T) to hybridize and form hydrogen bonds. This ...vectors or cloning arms with overhanging 3´ deoxythymidine (T) residues that are covalently linked to ...

...Carballosa, et al, (2015) eNeuro, March/April 2015, 2(2) e0001-15.2015. Pubmed. Resources: http://openspim.org...orthogonal-plane fluorescence optical sectioning (OPFOS) (2). Arne Voie, David Burns and Francis Spelman focused...in LSFM. In LSFM the laser light sheet, typically 2-6  microns, illuminates only one thin plane of the...(10).  He used a solution of benzyl alcohol and methyl salicylate which was later modified by others to...subpopulation of neurons are brightly labeled with GFP (e.g. Thy1-YFP-H mice).  However, we recently developed AAV...

...the addition of Cre recombinase (Figure 2). Figure 2: A knockout targeting vector designed to... through an experiment designed to knock out exon 2 of a given gene.   Figure 1: A knockout targeting...For recombination to occur in a cell, a minimum of 2 kb of sequence homology is required, but 6 to 14 kb...construct to your target cells. After recombination, exon 2 of the targeted gene will be removed from the chromosome...the GFP/NeoR cassette is inserted in place of exon 2. Thus, the targeted gene is disrupted (knocked out...recombination event will remove NeoR only and leave exon 2 floxed, as seen in line 4 of Figure 4. Since the loxP...genome, a negative selection marker like the HSV thymidine kinase (HSV-tk) is included just outside one of...

... constructed 2 versions of this reporter to target the K9 (pcDNA3-K9 histone methylation reporter) and... 525.7570 (2015): 533-537. PubMed PMID: 26352475. 2. Bygren, Lars O., et al. "Change in paternal grandmothers...in their DNA methylation patterns, either large increases or decreases. Hypermethylation of CpG islands...hybridization, methylation-sensitive restriction enzyme digestion, DNA adenine methyltransferase identification...linker is a methyllysine binding domain (chromodomain), which binds selectively to lysine-methylated peptides...the results. When methylation occurs at the histone-derived peptide, the methyllysine binding domain causes...to recognise lysine 27 methylation. When tested in living cells where methylation of these sites was controlled...

...generate 2 possible products: 1) a partially dsDNA duplex with single-stranded 3’ overhangs or 2) if the...recombineering system has three components (Figure 1): 1) Exo, 2) Beta, and 3) Gam. All three are required for recombineering...generic lambda red recombineering experiment (Figure 2). In the following sections, key steps that differ...confirmation of recombinant clones.    Figure 2: Overview of using Lambda Red recombineering system...pairs of homology to the targeted insert site (Figure 2, top).  These primers are typically ~70 nucleotides...bacteria that has key MMR proteins knocked out or 2) specially design ssDNA oligos to avoid MMR: 1) E....will have more unintended changes to their genomes. 2) Designing ssDNA oligos that avoid MMR activation:...