...about pooled library screening, read our recent blog post on Genome-wide Screening Using CRISPR/Cas9. If... population of cells as part of pooled library screens (1,2). The human and mouse GeCKO libraries, targeting...lentiCRISPR v2 is key to its efficient use in screening experiments; large amounts of virus are required...you get full library coverage in pooled library screens. Availability and Use LentiCRISPR v2 is available...Ophir, et al. "Genome-scale CRISPR-Cas9 knockout screening in human cells." Science 343.6166 (2014): 84-87... vectors and genome-wide libraries for CRISPR screening." Nature methods 11.8 (2014): 783-784. PubMed ...

...methodology of software development called Scrum. The Scrum framework is based on an iterative and collaborative...team because we are using the Scrum framework or we are using the Scrum framework because we are highly...collaborative approach to development. Scrum teams plan and break up work into small, well defined chunks...needs of the teams at Addgene. Another advantage of Scrum is that it provides transparency to everyone involved... on a new feature. We start with a story that describes the feature and what the criteria is for accepting...that the clarifications lead to the story being scrapped or broken up into smaller stories. Since this ...

...of customizable DNA-binding domains based on transcription-activator-like effectors (TALEs) as anchors ... thaliana. The results are light-inducible transcriptional effectors (LITEs) designed to bind specific.... Genes under LITE control showed a rise in transcription in as little as 30 minutes after light stimulation...News & Views Andreas Möglich and Peter Hegemann described several advantages of the new system: "First,... universally abundant. Second, induction of transcription occurs within minutes of light exposure. Third...really flexible and modular,” Konermann said. “Transcriptional activators are easy to expand or combine with...al. "Optical control of mammalian endogenous transcription and epigenetic states." Nature. 500, 472–476...

...quantify RNA transcription by RNA Polymerase III. By expressing Corn under promoters for transcripts uniquely...tagging RNA sequences with an aptamer and RNA transcription by placing the aptamer downstream of a known... Addgene’s Plasmids 101: Aptamer Fluorophores describes the enrichment process used to evaluate oligonucleotides... of colors, so do aptamers! Corn was recently described in 2017 (Song et al., 2017). Corn naturally dimerizes...uniquely transcribed by RNA Polymerase III (5S, tRNA and U6) and measuring Corn fluorescence by flow cytometry... Red and orange fluorescent aptamers Corn was described in the same study as Red Broccoli and Orange Broccoli...Wenjiao, et al. "Imaging RNA polymerase III transcription using a photostable RNA–fluorophore complex....

... friends on the Cambridge 2010 iGEM team that describes the basics of Gibson assembly as a parody of “...assembly The Gibson assembly technique was first described by Dr. Daniel Gibson and colleagues at the J. ...typically fairly high, so there is usually no need to screen a large amount of colonies. Excluding the time ...combined with the popular CRISPR technology and is described in a recent publication by the Lockey lab (Wang...followed by the standard Gibson assembly techniques described above, this resulted in the direct and seamless...using Gibson Assembly along with CRISPR was also described recently (Jiang et al. 2015), in which very large...challenging. Other homology based technologies We've described Sequence and Ligation Independent Cloning (SLIC...

... Figure 1: Initial steps in the CRISPR-pooled screening using sgRNAiBAR. Image from Zhu et al., 2019.  ... Peking University has developed a new CRISPR screening method that makes use of guide RNAs with internal...false-positive rate discovery rate, conventional CRISPR screens require a low multiplicity of infection (MOI) during... annotated human gene, it enables genome-wide screening with a high MOI, which is beneficial for a low... calcineurin dynamics and can serve as a drug-screening platform to identify new antifungals that target... assay, and are not suited to high throughput screening, calcineurin dynamics measurements, or monitoring...Sabrina Büttner’s lab designed a calcineurin transcriptional reporter consisting of a quickly-degraded destabilized...

...from one that represses transcription to one that activates transcription.However, this system lacks spatio-temporal...barbara_synbio) May 13, 2021 The original AraC transcriptional regulator Their system is based on the AraC...AraC protein that controls transcription from the PBAD promoter. “AraC is widely used in synthetic biology...It relies on the sugar arabinose to activate transcription by causing conformation changes in the AraC ...domains of the BLADE dimerize and activates transcription. The team created two constructs, among others...with AraC,” says Di Ventura. BLADE activates transcription with the flick of a light switch and shuts off...

...the transcribed gene and its 5’ UTR. Various self-cleaving hammerhead ribozyme parts are described in ...of an RSM is defined by the sequence within a prescribed region of DNA termed the “register”. Each chemical...registers within a sample using a metric that we describe in the Materials and Methods section of our paper...sensors do not necessarily have to be based on transcriptional regulation, as long as they can control recombinase...recombinase activity. For example, post-transcriptional recombinase activation via RNA toehold riboregulation...to read states by qPCR or Sanger sequencing as described in the Material and methods section of our paper... Multiple large serine recombinases have been described in the literature (5, 6) and may be substituted...

...lentiviral packaging methods. The three components described above (envelope, packaging, and transfer) are ...nomenclature of the plasmid backbone is meant to be descriptive of the components found within the backbone. ... Pol Precursor protein containing Reverse Transcriptase and Integrase components. Transfer cPPT... WPRE Woodchuck hepatitis virus post‐transcriptional regulatory element; sequence that stimulates...necessary for activation of viral genomic RNA transcription. R; Repeat region found within both the 5' and... 5’ LTR Acts as an RNA pol II promoter. The transcript begins, by definition, at the beginning of R,...rest of the provirus. 3’ LTR Terminates transcription started by 5' LTR by the addition of a poly ...

...972.e23 Screen identifies bromodomain protein ZMYND8 in chromatin recognition of transcription-associated...times have you looked at a diagram depicting transcription, or DNA repair, or replication, or any number...researching enhancer elements that control the transcription of a gene a megabase away, or looking at the...plasmids containing the indicated gene, and you can subscribe to these pages to learn when Addgene receives ... articles reporting chromatin-related research describe the production of plasmids that might be useful...colleagues and guest bloggers have written posts describing new methods and tools for studying chromatin ...

... lentiviral vector that contains a scrambled shRNA. This scrambled shRNA is used as a negative control...sequence (pSpCas9(BB)). The 2A-GFP allows for screening of transfected cells. Find pSpCas9(BB)-2A-GFP ...virus, making it ideal for CRISPR pooled library screening experiments. Find lentiCRISPR v2.(If you’ve peeked...envelope expressing plasmid pMD2.G. Find pMDLg/pRPE. scramble shRNA - This plasmid from David Sabatini’s lab...control for mammalian RNAi experiments.  Find the scrambled shRNA vector. pLKO.1 - TRC cloning vector - This...the necessary elements for packaging, reverse transcription, and integration needed for the production of...

...fused to a transcriptional repressor to your gene of interest. It will inhibit transcription instead of...stranded RNA molecules that degrade target mRNA transcripts. When transiently introduced, they can deplete...that, too! CRISPR activators (CRISPRa) are transcriptional activators fused to a dead Cas9. The system...tetracycline or one of its analogs will induce these transcriptional changes, and expression is tunable by titration...expression goes back to baseline) and occurs at the transcriptional level.     Fig. 2 Tetracycline...post-translational level, unlike most of the other mechanisms described. Fig. 3 dTag mechanism of protein degradation...

...xCas9 for transcriptional regulation and base editing Hu et al. also engineered transcriptional activators...activating transcription. Sites containing NG, GAA, or GAT PAMs averaged 56-91% transcriptional activation...subunit ω. This construct is designed to activate transcription from the accessory plasmid (AP) encoding phage...could recognize a library PAM, it would induce transcription of gene III, permitting phage propagation and... of xCas9 3.6 and 3.7 shows that we have only scratched the surface of modifications that can be made ...

...Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening, a protocol paper by the Zhang lab...library has inserts of different sizes (such as a transcription factor overexpression library) or is prone to...Re-transformation of the SPLINTR Libraries (PDF, 401 KB) Screening Zhang Lab - Multiplexed Overexpression of Regulatory...et al. Nat Protoc. 2017 with modifications Screening pCMV-Rep78/68 Scanning Saturation Mutagenesis...Amplification and Sequencing Protocol (DOCX, 32 KB) Screening Rinehart Lab Iterative Synthetically Phosphorylated...More resources on the Addgene blog Genome-Wide Screening Using CRISPR Preparing Pooled Libraries NGS Quality...

...open reading frame (ORF) library to screen PPIs among 450 transcription factors in live fruit flies. Although..., kinase signaling, integrin signaling, and transcription factor interactions (Kerppola, 2006). One of...interactions between basic leucine zipper and Rel transcription factors (Hu et al., 2002). This would be more...large number of constructs for high-throughput screening of PPIs. For example, Bischof et al. (2018) generated...organisms lags behind. BiFC can also be used to screen for small-molecule inhibitors of a particular PPI...quantification (Kerppola, 2006, 2013). These are described in more detail below: Fusion protein construction...

...recent resurgence of “Do-It-Yourself Science” described in Chapter 3, and of course the rapidly changing... science.”  The three chapters in this section describe the challenges of trusting science in a “fake ... of the book, called simply “Old Science,” he describes the now infamous tale of Rosalind Franklin and...changed significantly over the last decade. He describes the detection of gravitational waves--a story ...collaboration from scientists around the world. The paper describing the waves’ detection had over 1,000 authors. ...This last section of the book, Future Science, describes the author’s thoughts on science’s biggest challenges... doesn’t need to. The problems and challenges described in The State of Science have not changed in “COVID...

...CRISPR screen to disrupt these enhancers. The third and final step is to conduct our CRISPR screen. We start...stem cells, and conclude with a targeted CRISPR screen that can disrupt these enhancers. A real-world ...“GPS for proteins”) for NANOG, an essential transcription factor in embryonic stem cells (ESCs). Your ...that have NANOG binding sites 2. Design a CRISPR screen to target and disrupt the NANOG enhancers 3. CRISPR...you’re interested in designing your own CRISPR screens for enhancers, check out the Appendix. Conclusion...established with Bowtie2 using the parameters first described in Kearns et al.: bowtie2 -f -x HG19_GENOME --...

...after the piñata burst, value was subjectively ascribed relative to the basic needs of each individual... few prior publications to go on, a submitted manuscript could only have been viewed as a significant ...There are 8 major categories found in the table, describing everything from basic to advanced functionality...the user is expected to interact with the tool (a screen shot and overview of the table can be found above...Software design elements that fall into this category describe how the user is expected to interact with the ...require working knowledge of the command line or scripting. Input flexibility: Software tools require some...re engaged in heavy CRISPR design (I’m talking screens and/or very large genomes), then it is worthwhile...

...reverse transcription. This is then amplified by PCR using Taq Polymerase and then transcribed to provide...Cas12a activity gets activated. This unleashes indiscriminate ssDNA cleavage activity which cleaves the supplied...amplified. RPA reactions also contain reverse transcriptase to produce cDNA. Isothermal amplification is...thermocyclers. This is followed by in vitro transcription and Cas13 is then used to detect the amplified...designed, each binding to one strand of the reverse-transcribed target sequence that are present close to the...fluorescence upon cleavage. Highlights: Reverse-transcribed RNA samples are amplified by PCR instead of RPA...

... They use a unique and powerful high-throughput screen to select variants based on performance metrics...: JEDI-2P was identified from a high-throughput screen, selected for increased voltage response, brightness...a new preprint, Jared Toettcher and colleagues describe pYtags, a modular system for monitoring specific...developed using a high-throughput multiparameter screen (Koveal et al. 2022). Lactate is a key metabolite...cellular concentrations of lactate. The microfluidic screening platform developed here is also a promising approach...Figure 6: A) Schematic of BeadScan platform for screening candidate biosensor variants for performance under...Koveal D, et al. A high-throughput multiparameter screen for accelerated development and optimization of...