...researchers to study aspects of the EBOV replication cycle under BSL-2 conditions (4). What is a minigenome...minigenome replication and reporter gene transcription by the EBOV polymerase (reviewed in 2, 5). The minigenome...template for replication and transcription, as it would for a full-length viral genome (Figure 2). While it...nonsegmented negative sense RNA viruses (reviewed in 2, 3). Other important components of the Zaire ebola...500 ng NP, 500 ng VP35, 100 ng VP30, 100 ng L, and 2 μg minigenome plasmid per well (in a 6 well plate ...PubMed PMID: 9847328. PubMed Central PMCID: PMC103829. 2. Conzelmann, K. K. "Reverse genetics of mononegavirales...Technical Tips). Minigenome replication and transcription In order to replicate the minigenome, the viral...

...Figure 2: Effects of (A) double and (B) single base pair gRNA-target mismatches on modification by AsCpf1...class 2 CRISPR-Cas system." Cell (2015). PubMed PMID: 26422227. Find plasmids from this publication at ...Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Figure from Zetsche et al., 2015...2015. How was Cpf1 found and tested? Class 2 CRISPR systems, including the type II Cas9-based system...employed by class 1 systems. A putative new class 2 nuclease, Cpf1 (CRISPR from Prevotella and Francisella...at Addgene. 2. Zetsche, Bernd, et al. "Multiplex Gene Editing by CRISPR–Cpf1 Using a Single crRNA Array... this publication at Addgene. Makarova, Kira S., et al. "An updated evolutionary classification of CRISPR-Cas...

...Originally published Dec 7, 2017 and updated Jul 2, 2020. Promoters may be the star of gene regulation...sequence targeted in the experiment. Figure 2: Comparing CAPTURE and CAPTURE 2.0.     Original...cell line expressing 1) FLAG-biotin-tagged dCas9, 2) BirA, and 3) one or more gRNAs Two separate lentiviruses...delivering 1) a BioTAP-tagged dCas9 and an eGFP tag, and 2) gRNAs Number of loci targeted One Many Sample...identified and the level of on-target enrichment. 2. Determining the spatial and hierarchical organization...Cytogenetics, 11. https://doi.org/10.1186/s13039-018-0368-2  Liu X, Zhang Y, Chen Y, Li M, Zhou F, Li K, Cao H... gene’s transcription. Sometimes enhancers are located thousands of base pairs away from the gene they...

...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...helps us to locate interesting addresses in the genome. Let’s now examine how we can locate powerful stretches..., start, and stop. For instance (chr3, 1, 10) indicates a stretch of DNA at the very beginning of the ...

...University of Manitoba due to their Biosafety Level-2 or 2+. Please Note: Despite the fact that AAVs themselves...important factors related to location. Location (1): What tissue? Location (2): Where in the tissue/which...long and stable expression (up to 9 months in mice [2]). Low Immune Response: Compared to other viral vectors...may be sufficient to overcome the transport issues. 2. Failure to plan (for storage) Is planning to fail...Find Ready-to-Use Retrograde AAV Preps at Addgene 2) Comparison of genetically engineered receptors (i.e...Therapy 15.10 (2007): 1740-1743. PubMed PMID: 17882145. 2. Zincarelli, Carmela, et al. "Analysis of AAV serotypes... tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain." PloS one 8.9 (...

... can be used to diagnose the presence of SARS-CoV-2 RNA, but inadequate access to reagents and equipment...named this system SHERLOCKv2 for SHERLOCK version 2. Here are some of the improvements: SHERLOCKv2 uses... a protocol for using SHERLOCK to detect SARS-CoV-2 RNA. The test is started using RNA purified from patient...recently adapted the DETECTR platform to detect SARS-CoV-2 using a lateral flow strip format. They published ...CY (2020) CRISPR–Cas12-based detection of SARS-CoV-2. Nature Biotechnology. https://doi.org/10.1038/s41587... and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems. Molecular Cell 60:385–397 . https...Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell 163:759–771 . https://doi....

...that have been developed into retroviral vectors and 2.) a subclass of retroviruses called the lentiviruses...effectively. Retroviral structure   Figure 2: Structure of a retrovirus. Image re-used from Dong...Dong & Kantor, 2021 under CC-BY license. Figure 2 gives a detailed view of the structure of a retroviral...transfer of RT, which would actually occur in step 2 during uncoating. After uncoating, reverse transcription...one of the two strands’ DNA counterparts from step 2 will be integrated by the viral integrase protein ... Air. Environmental Science & Technology Letters, 2(4), 84. https://doi.org/10.1021/ACS.ESTLETT.5B00050...retroviruses ” (Cullen, 1992) because of their more complicated genomes. Here, we will mostly use the gamma-retroviruses...

...and Amy tell us a little about themselves. Chapter 2: What is Science in the News (SITN)? - SITN is a graduate... to be crazy complicated! Listen to the full podcast for more great science communication tips or listen...tons of ways you can get involved in science communication. In this second half of our conversation with...many things you can do start your own science communication student group and get more involved with your... graduate student group with the goal of communicating science to the general public and training the ...the next generation of science communicators. In this chapter we learn about some of their events - science... peer-to-peer feedback in effective science communication. Chapter 7: The Goal of the SITN Podcast - Vini...

...fluorescent state make them useful for 2-color imaging with green PA-FPs. Table 2: Properties of Selected Reversible...conventional FP imaging. PA-FPs are divided into 2 main categories - those with irreversible and reversible photoactivation... forms of the chromophore which contribute to the 2 peaks of wtGFP’s excitation spectrum- a major 397 ... / N99K / S148L / I165V / Q167P / L169V / I203R) (2) and PA-mRFP1(S146H / I161V / I197H) (3) were derived...with two distinct wavelengths of light (see Table 2). The best known reversible PA-FP is the naturally... above background. The majority of PA-FPs come in 2 flavours- monomeric and tetrameric. Tetrameric PA-...most PA-FPs, but, in 2015, the Looger Lab designed 2 variants of EosFP that can withstand these conditions...

...Golden Gate Assembly is commonly used to assemble 2–10 DNA fragments in a single “one-pot” reaction to...the transformants onto the selection plate. Figure 2 shows representative transformation plates obtained...fidelity studies conducted by Potapov et al. at NEB (1, 2). While T4 DNA Ligase, the mainstay of most biotechnological... (percent correct) Calculated colony totals Per 2 μl assembly reaction Per full assembly reaction** ...calculated yields from entire outgrowth built from 2 µl of the assembly reaction, and from the entire assembly... bias profiling." bioRxiv (2018): 322297. 2. Potapov, Vladimir, et al. "A single-molecule sequencing assay...upon growth on LB/Cam/X-gal/IPTG agar plates, indicating successful reconstruction of the coding sequence...

...resulting from non-homologous end joining (NHEJ) (2-5), depending on your desired genome edit. Since exploitation...each sample can be quickly calculated (details in [2]) and can be used to quantify rates of mutation in... have been reported for mismatch nuclease assays (2, 6, 7). Ability to distinguish between mono-allelic...assays, in addition to testing their performance (2). Remember that although digital PCR is a great screening...PMID: 24509632. PubMed Central PMCID: PMC4063274. 2. Findlay, S. D., Vincent, K. M., Berman, J. R. & Postovit...a gene of interest is one of the most common applications of precision genome editing, the remainder of...single molecule resolution of digital PCR allows classification of individual target molecules based on their...

... H. J. (2009). Rats! Disease Models & Mechanisms, 2(5–6), 206–210. https://doi.org/10.1242/dmm.002733 ...studying genetic modification for their ease of manipulation and their ability to be replicated. Because E...., 2004). Additionally, breakthroughs in gene modification tools and methods such as CRISPR have vastly...Escherichia coli as a Model Organism and Its Application in Biotechnology. In Escherichia coli - Recent...Physiology, Pathogenesis and Biotechnological Applications. InTech. https://doi.org/10.5772/67306 Koornneef... bacteria with superior performance. Nature Communications, 9(1). https://doi.org/10.1038/s41467-018-04899...

...all of the following modifications: 1) increased concentration of Taq polymerase, 2) increased PCR extension... that results in a polymerase error rate of up to 2% per nucleotide position. The “sloppy” PCR reaction...to synthesize a complementary mutant strand (step 2 and 3). Then the uracil-labeled template strand is...restrict site matter? Most of the time no, but there are 2 instances where it does. 1) When the backbone vector...ExoIII. See the below figure for an example like this. 2) When your gene of interest has a BbvCI site(s). If...saturation mutagenesis method are 1) ease of use, 2) time savings, and 3) ability to perform single site...PMID: 23766324. PubMed Central PMCID: PMC4409680. 2. Mccullum, E. O., Williams, B. A., Zhang, J., & Chaput...

...is funded have been well documented over the past 2 years (Alberts et al, PNAS 2014; Taylor, eLife, 2016...PMID: 24733905. Pubmed Central PMCID: PMC4000813. 2. Taylor, Justin Q., et al. “Avoiding a lost generation...Riglar Advocating for Science Symposium and Workshop 2016 – Sept 16-17 MIT, Boston The Advocating for Science...futureofresearch.org/advocating-for-science-boston-2016/. Sign Up For the Advocating for Science Symposium...Director of Media Communications, Harvard University Ray Howell -Founder of Howell Communications Philip Brenner...union dues. Time will tell the extent of the implications of this ruling and whether graduate students...labor laws, rather than as trainees in a special category, as has been the ‘labor-of-love’ philosophy of...

...illustrated in the schematic below.   Figure 2: gRNA target sequences (colored rectangles) are cloned... plasmids: This plasmid set allows you to express 2-4 gRNAs, with four being the ideal number. First you...the total number of gRNAs you wish to clone, from 2-7. For example, if you’re expressing 4 gRNAs, you’...gRNAs into spectinomycin-resistant plasmids pX330S-2 to pX330S-(last gRNA number). The 5’ most gRNA is ...Multiplexing Plasmids: These plasmids allow you to assemble 2-4 gRNAs through Golden Gate or Gibson Assembly. gRNAs...Multiplex Plasmids: These plasmids allow expression of 2-5 gRNAs in zebrafish. Custom destination vectors are...and a subsequent curing protocol that requires only 2-3 hours incubation. Kondo Lab multiplexed base editing...

... PMC3918502. Find plasmids from this publication at Addgene. 2. Ma, Hanhui, et al. “Multicolor CRISPR...two genes over time, at a range of distances from 2-75 Mbp. Labeling multiple, intrachromosomal loci Building...successfully resolved loci with physical map distances of 2 and 75 Mbp, with the calculated fluorescent distances...established physical map. In comparing pairs of targets ~2 Mbp apart, they noticed that they could evaluate the...dCas9 supplied by a separate vector. Figure 2: CRISPRainbow generates 6 unique colors and a white... every genomic locus, and to do so in vivo. Catalytically dead Cas9 (dCas9) cannot induce gene editing...Cas9 ortholog, and the target sequence must be located adjacent to that PAM. To scale up fluorescent CRISPR...

... of 2 components: 1) an SB transposase, the enzyme required for catalysis of transposition; and 2) a transposon... gene (red) for expressing the transposase enzyme.2.) The transposase enzyme is expressed (red stars) ... 2015 Feb 4. doi: 10.1002/biot.201400821. PubMed. 2. Mates L, et al. Molecular evolution of a novel hyperactive... Specifically, viruses are able to infect and replicate in host cells, but they are susceptible to cells... though not all, of these defenses. For some applications of genome engineering - such as certain forms...to integrate. Additionally, the TA sequence is duplicated near the gene of interest insertion site.   The...containing the gene-expression cassette that can translocate within the genome. The Sleeping Beauty transposase...

...TUs. Level 2: Assembly of multiple Level 1 TUs into multigene constructs. Figure 2: The three ...can be queried by testing any number in the Level 2 assembly. These pathways can require a lot of optimization... of multigene constructs. PLoS One. 2011 Feb 18;6(2):e16765. doi: 10.1371/journal.pone.0016765.  Hahn,... (2020). https://doi.org/10.1186/s12870-020-02388-2 Resources on Addgene.org Addgene’s Modular Cloning...most obvious example of an individual part, this category also includes promoters, UTRs, terminators, etc...multi-purpose it can be. Let’s review a list of MoClo applications. Genetic circuits Most things in biology are...performing production and subsequent necessary modification of these chemical products. The flexible nature...

...estimate transformation efficiency (Figure 2).    Figure 2: Example of a serial dilution to estimate...ask for an amplification protocol with your deposit. This is because repeated amplifications of pooled ... an amplification protocol developed for each individual pooled library; generalized amplification protocols...Plasmids Variable Region Size DNA per Amplification Amplification Type Link to Protocol CRISPR Knockout... this blog, we’ll discuss how to develop an amplification protocol for your pooled library deposit. When...to amplify the sample for further downstream applications. As individual libraries can vary significantly... to consider when creating a pooled library amplification protocol. Created with BioRender.com.   ...

... in the brain region you wish to stimulate 2) Fabrication and implantation of the optical fiber into .../nrn3171. PMID: 22430017. 2. Fenno L, et al. The development and application of optogenetics. Annual review...commonly used in optogenetics: 1) electrophysiology and 2) behavioral experiments. I’m not an electrophysiologist...thoroughly elsewhere (here and here for instance) [2], [3]. Karl Deisseroth of Stanford University , one...reviews for a discussion of the different variants [1, 2, 4]. And, of course, the best way to learn about the...monitor expression at various time points (e.g. every 1-2 weeks over 8 weeks) in order to determine the optimal...analysis of microbial opsins. Nature methods. 2012;9(2):159-72. doi: 10.1038/nmeth.1808. PMID: 22179551; ...