... ready for experimentation. In 2014, several manuscripts published in Genetics detailed approaches in ...variable amounts of experimental manipulation and screening.  A co-selection strategy for CRISPR editing in... with inefficient sgRNAs in my initial direct screening efforts, which were similar to the approach detailed...low efficiency (0.13%) and laborious handling (screening 768 F1 animals). Recovering rare editing events...conditional-lethal mutation to identify edits with minimal screening effort. Selecting for repair of a lethal mutation...to identify a single 2xFLAG knock-in by direct screening. The center images depict the power of lethal ...parental animal, are rescued progeny, which makes screening extremely rapid. This stringent selection allowed...

...isolated and screened. Depending on the marker, traps can be used as high-throughput screens to introduce...Each trap uses a slightly different approach (described in detail below), but fundamentally the reporter...models. Why traps are useful Traditional genetic screens employ various on/off switches to measure the function...circumstances, trap methods may be the preferred screening option. Promoter traps Promoter trap systems ...integrate within an exon of a gene to generate a transcriptional fusion. This may disrupt the endogenous function...spliced into the gene product resulting in a transcriptional fusion, much like promoter traps. While this...likely disrupt the gene it monitors due to transcriptional fusion.   Examples of promoter, enhancer...

...large scale functional screens. For more information on these new CRISPR screening tools, see our CRISPR... Lentiviral CRISPR libraries for knockout screening New systems have been developed and deposited ...CRISPR-Cas technology to perform genome-wide knockout screens. These vectors and sgRNA libraries expand upon ...CRISPR Libraries Enable Genome-Scale, Knockout Screening. Also, check out our updated CRISPR-Cas resources...minimizing confounding secondary effects? An approach described as “knocksideways” (a British idiom for “taking...Chrimson Through de novo sequencing of 127 algal transcriptomes, as well as further optimization through engineering...two-color optical excitation of neurons. The paper describes five previously unknown channelrhodopsins from...

...Direct reprogramming describes the process where differentiated cells are turned into a cell type of ...cell technologies, such as single cell RNA-seq (scRNA-seq), now allows scientists to identify the gene...cells that successfully reprogram. Unfortunately scRNA-seq only captures the gene expression of cells at...identifiers act like a timestamp and help demultiplex the scRNA-seq data and build multi-generational cell lineage...reprogrammed into iEPs by retroviral expression of transcription factors Foxa1 and Hnf4α, a process that like...reprogramming (CellTagMEF), at the end of the three-day transcription factor reprogramming treatment (CellTagD3), ...three days, a sample of cells was collected for scRNA-seq analysis and the remaining cells were maintained...

...Detect Zika Virus The RNA-based toehold switches described in this post are engineered to bind RNA sequences...Boston University School of Medicine, this post describes a tool you can use to study basic virus biology...sequences required for viral replication and transcription as well as reporter genes. To learn how you ...researcher in the Silver Lab at Harvard University, describes R-bodies, “force generating protein machines that... Connie Cepko’s Lab at Harvard Medical School describes how AAVs function and indicates important things...CombiGEM and CRISPR for Combinatorial Genetic Screening CombiGEM is a technology that can be used to quickly...barcode small libraries of CRISPR gRNAs to use for screening experiments. Get the full details on how to make...

...commonly used scripts: If you edit a general purpose script for a single use, save the script as originalName_descriptionOfEdit...be in the scripts that you run. Rather, this is where you outline your logic and the scripts and input...immediately write a text document with a brief description of the directory contents, at a minimum. This...for all of your analyses. The same is true for scripts. Naming conventions Give every experiment a number... remains in tact! Take notes Comment in your scripts: Comments on what a given line of code does are...

...effort into our quality control (QC) processes to screen for and reduce these errors. These processes allow...types of data for each of their plasmids, from descriptions of key components of the plasmids to partial...process ensures that any potential sequence discrepancies can be caught prior to distribution. Catching...issue? At Addgene, a QC issue is defined as a discrepancy that we believe may affect the function of the...decisions when analyzing the wide variety of discrepancies encountered in plasmids deposited to Addgene...resolve. Sequencing and assembly issues Sequence discrepancies are not always a result of mutations in the ...will contact the depositing lab to review the discrepancies. Often, the identified issues will not affect...

...with a publication in Science describing the development of “transcriptors,” transistor-like digital genetic...cellular computing. These logic gates built from transcriptors are now available at Addgene through the Biobrick...kit comprised of well-characterized bacterial transcription and translation initiation elements. Open science...with the central dogma - replication of DNA, transcription of DNA to make RNA, translation of RNA to make... and observed that we humans find it useful to describe and interact with the world using words like OR...The future of synthetic biology Addgene: You've described the pieces in these kits as the beginnings of ...systems today who are building 10-gene systems from scratch. (They may be building genomes, but that's just...

...genome-wide screens, introducing bias into all work based on these and any other CRISPR screens using the..., race. Many researchers do large-scale CRISPR screens in cancer cell lines, looking to uncover genetic...targets and related biomarkers . Data from such screens are often uploaded to the Cancer Dependency Map...Boehm labs pulled data from more than 1000 CRISPR screens in DepMap to see if they could identify the ancestry... American, Africa, or South Asian) used in the screens based on the cells’ germline genetic variations...about 90% of the cell lines used in DepMap CRISPR screens are of either European or East Asian ancestry, ...actually reference?  The CRISPR guides used in the screens they analyzed were designed to have a perfect match...

...dCas9 has focused on transcriptional activation using a fusion to a transcriptional activator such as VP64...repression of transcription through binding a promoter region to inhibit association of transcriptional activators...region In 2013, the Hodaka Fujii Laboratory first described a method to purify a specific genomic region using...locus of interest, the promoter region of the transcription factor IRF-1. Immunoprecipitation, using an ...plasmid. Experimental use Since the initial report describing CRISPR-mediated purification of a specific genomic...Tapscott Lab identified regulators of DUX4, a transcriptional regulator that when inappropriately expressed...CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes." Cell 154.2 (2013): 442-451. ...

...Molecular cloning requires some method of screening colonies for the presence of an insert. Traditionally...backbone. This type of primer pair is also great for screening clones created with the same backbone but that...is, allowing you to use the same primer pair to screen for the presence of many different inserts. The...test your colony PCR primers before using them to screen colonies. The best way to do this is by using your...insert and if it's in the plasmid Can be used to screen clones that were created with the same backbone... background, the more colonies you will need to screen.   Saving clones for later culture: At this point...expected results for the three primers previously described. When using insert-specific primers (1), positive...

...a plasmid kit designed to do exactly that. As described in the journal Science Signaling in December 2014... to each to make the library usable for pooled screens. Applications of the cancer pathways ORFs kit Wood...and his colleagues developed the kit for use in screening for pathways that drive resistance to drugs in...the only way to use the kit. “You could use it to screen for pathways that confer any phenotype of interest...drug resistance,” he said. “It could [be used to screen for] morphological features, migration, or survival...use our libraries to do things - sometimes drug screens and sometimes totally different experiments we ...Tracing Read How CRISPR Can Be Used in Genome-Wide Screening Browse 28 Hot Plasmid Technologies from 2015 ...

...targeting dCas9 to transcriptional start sites was sufficient to repress transcription by blocking its initiation...desired edit adjoined to the sgRNA, a reverse transcriptase, and a nCas9. While this system has been shown...Prime editor engaging target DNA. The reverse transcriptase polymerizes DNA onto the target strand based...been exploited to localize factors such as transcriptional activators and repressors to target genes. ...repression requires targeting dCas9 along with transcriptional repressors to the promoter region of the gene...associated box (KRAB) is a naturally occurring transcriptional repressor domain which is frequently fused ...activators consists of dCas9 fused to a single transcriptional activation domain - VP64.  Later generations...

...assays for your screen result may or may not actually help. Ultimately, if you cannot describe the likely ...of experiments or a laundry list of molecules to screen has unclear utility. You cannot predict what will...cannot either. Therefore, you need to be able to describe experimental approaches in terms of the question...approach, anticipated outcomes, and preferably a description of the payoff if the aim is successful. Note ...the aims and clarify what type of aim you are describing. It's easy to be overly ambitious – a common ...distinguish mechanistic questions from open ended screens and data collection. Organize your research vision...accomplished in 5-10 years? You need to be able to describe what success looks like. If you are successful...

...hot plasmids segment on the Addgene Podcast. Subscribe to the Addgene blog to get all the latest hot ...pluripotent stem cells) through the expression of transcription factors OCT3/4, SOX2, KLF4, and c-MYC is the... method for generating iPSCs to express the transcription factors from a polycistronic cassette. However...polycistronic cassettes and discovered reprogramming discrepancies, which they traced to a 9 amino acid N-terminal... did not lead to the observed reprogramming discrepancies. The polycistronic piggyBac transposon vectors...the RFPs developed from natural ancestors), and screened through libraries of new RFP variants for increased...beautifully demonstrate light-dependent Gal4 transcriptional activation and Cre recombination. Find PhoCl...

...Dr. Theodor Escherich, the scientist who first described them in 1885. E. coli are mainly found in the ...mutations found in E.coli strains Gene(s) Description Functional Consequence dam DNA adenine methylase...methylate DNA lac Lac operon mutations Blue/white screening of clones   lacIq lac repressor overproduced...derivative. General cloning and storage, blue/white screening, leucine auxotroph. F- endA1 recA1 galE15 galK16...cloning and storage of common plasmids, blue/white screening. F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR...General cloning and plasmid maintenance, blue/white screening, partly restriction-deficient; good strain for...derivative. General cloning and storage, blue/white screening. F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74...

...our AAV preparations. We recently published a description of the VGS process and use cases in Human Gene...ligation and tagmentation preparation approaches described previously require a preliminary double stranding...addition, we wrote an open-source custom Python script that uses the VGS data to determine the percent...recombination present in Cre-dependent vectors and a script that can confirm the serotype of AAV preparations...use this to our advantage by designing a Python script that looks for pre-defined serotype specific signature...signature sequences in the data. Unlike previously described thermodynamics-based methods of serotype confirmation...parallel and provide serotype data in minutes and the scripts  can be easily customized to detect other sequences...

...is the rising use of preprints, or scientific manuscripts shared by the authors on a public server independent... their work publicly and immediately once the manuscript is drafted. Preprints were one of the key routes...same time as, or slightly before, submitting the manuscript to a journal for formal peer review. Some organizations...unpublished results at a conference? Post your working manuscript as a preprint too and you can share all the details... use your work but don’t think they’ll have subscriptions to the specialized journals in your field? Post...only way to rigorously determine or ensure a manuscript’s quality. But even journal-based peer review ...giving authors the opportunity to strengthen the manuscript prior to submission. Preprints at Addgene Preprints...

...contains a detailed description of the experimental conditions.     Figure 4: A screenshot of the materials...antibody worked under the conditions described above, and a short description of how the antibody performed....the submitted application, along with a brief description of the experiment. If that data was included ...conditions in which the data was generated, you can scroll down the page to the materials and methods section...The submission process is easy, with simple, descriptive fields, drop-down boxes when appropriate, and...

...reverse-transcribed DNA into the host cell’s genome and then use cell machinery for transcription and translation...structural proteins expressed from a reverse-transcribed piece of DNA called a “provirus.” This DNA is...(or host cell) by the viral protein reverse transcriptase (RT). In the case of a natural virus, both the...the protein and the genetic code for reverse transcriptase are packaged (Wu et al., 1997) inside of the...viral vectors, only the protein for reverse transcriptase (RT) is present in the capsid, as the engineered...is a turn of phrase — “pseudotransduction” — to describe the direct protein transfer of RT, which would...during uncoating. After uncoating, reverse transcription follows the release of the two strands of viral...