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...of many complex cellular behaviors, such as differentiation or metabolism. Our attempts to manipulate these...cerevisiae with few techniques that are easily transferrable across host species or metabolic pathways. A...system for multidirectional control The CRISPR interference (CRISPRi) model consists of a catalytically .... cerevisiae by using three RNA programs to differentially alter the levels of five genes that encode ...sequentially rerouted the metabolic flux to generate differently pigmented derivatives of violacein. Using dCas9...that will still recognize guide RNA but with a different dCas9 binding module, alternative sets of transcriptional...pharmaceuticals. Follow him on Twitter @AdamChinFatt     References Qi, L. S. et al. Repurposing CRISPR as an RNA-guided...

...activation p300 acetyltransferase dCas9 fused to the catalytic domain of p300 acetyltransferase increases levels...transcription, also includes light-regulated histone methyltransferases and deacetylases. CRISPR and epigenetics ...delivery systems. Transcriptional repression DNA Methyltransferase 3 Alpha (DNMT3A) Vlatka Zoldoš’ lab has deposited...repression. Created with BioRender.com. DNA Methyltransferase MQ1 Margaret Goodell's lab has deposited pcDNA3.1...Q147L)-EGFP, a fusion of dCas9 to a small DNA methyltransferase from the prokaryote Mollicutes spiroplasma...Schwiesow contributed to updating this article. References Haldeman JM, Conway AE, Arlotto ME, et al (2018... Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers...

...they do arise. Happy CRISPR’ing! References and Resources   References Rabinowitz R, Offen D. Single-Base...cutting to occur. The RNA sequence and PAM site confer specificity of the nuclease; however, many Cas ...effects should impact your experimental design differently. Yes, it is always better to reduce off-target...mutation, off-targets must be quantified slightly differently. For a comprehensive approach, RNA-seq would ...to control for off targets as well as clonal differences. If you are performing CRISPR library screens... as a transfection or transduction control, a different gRNA would have distinct off-target sites, eliminating... there really aren’t any effective off-target reference controls. Off-targets and your experiment Every...

...For more context, exposition, and detail, please refer to the paper. Why might one be interested in state...register (Fig. 1C below). If attB-attP pairs from different recombinases are overlapping or nested on a register...that enters distinct states when treated with different combinations and orders of two recombinases. ...leveraging qPCR data from multiple primer pairs, one can infer the predominant state of registers within a sample...strong, directional terminators and promoters with buffer sequences on their 5’ and 3’ ends (for insulation... register. The purpose of this is to prevent interference between the transcribed gene and its 5’ UTR.... in biocomputing and biological circuits.     References 1. N. Roquet et al., Synthetic recombinase-based...

...by Hannah Dotson in Dec. 2022.  References and Resources References Gossen M, Bujard H. Tight control...CMV, EF1A, and SV40, are always active and thus referred to as constitutive promoters. Others are only ...In the presence of tetracycline (Tet), TetR preferentially binds Tet over the TetO elements, allowing ...hear some transcription factor and promoter pairs referred to as binary systems. Binary systems are genetic... One can therefore place GAL4 and GAL80 under different promoters and create sophisticated patterns of...wondering, “why is it necessary to have all 3 of the different binary systems above if they all behave very similarly...s00253-018-9485-4. Epub 2018 Nov 3. PMID: 30392122. Pfeiffer, Barret D., et al. “Refinement of Tools for Targeted...

...pooled libraries are single preparations of many different plasmids. Plasmids within a given library have...the same backbone, but they express or target different genes. Some libraries cover the majority of the... library from the Moffat lab has over 175,000 different gRNA-containing plasmids). Amplifying and Using...screen. If you need to amplify the library, please refer to the depositor’s protocol for the best results...conducting pooled library screens. Addgene does not offer next-generation sequencing services. For an NGS ...Genome-Scale, Knockout Screening Pooled CRISPR Libraries Offer Genome-Wide Control for Large-Scale Functional Screens...

...the liquid. Molecules absorb different wavelengths of light to different degrees and many molecules have...cuvettes. First you measure the absorbance of the buffer that the DNA is in. This is a “blank” and measures...the ladder. You then run samples of your DNA at different dilutions and quantify based on band intensities...elute and store your plasmid DNA Learn about the different grades of plasmid DNA preps Resources on Addgene.org...Addgene.org Visit the Molecular Biology Reference Find plasmids for your research Browse protocols for ... Jennifer Tsang...

...in 1983. The differences between gram positive and negative bacteria is due to differences in cell wall...incredibly helpful in visualizing differentiations between bacteria, leading to safer medicine and more successful...minoring in studio arts photography.  References and resources References Silhavy, TJ., Kahne, D. & Walker ...negative and positive bacteria are permeable to different classes of antibiotics. The classification system...

... the transcript. Different Cas orthologues and subtypes function somewhat differently and can be more ...Transcriptional repression (CRISPRi) in bacteria As RNA interference does not function in bacteria, most efforts ...promoters or direct gene knockout. In contrast, CRISPR offers a much more user-friendly way to modulate gene ...Peters, Oren Rosenberg, and Carol Gross used two different genetic systems, targeting either Gram-negative...bacterial species (Peters et al., 2019). The systems differ on how exactly the CRISPRi locus is introduced ...Clostridium difficile, could be targeted and preferentially depleted from gut. While still far from the...the BE3 that further improves editing rates. References Bikard D, Euler CW, Jiang W, Nussenzweig PM, ...

...updated by Jennifer Tsang in August 2020 and by Emily P. Bentley in September 2025. References and Resources...for resolving DNA double-strand breaks (DSBs) preferred by researchers. Compared to non-homologous end...APOBEC3G displayed less predictable sequence preferences.  The Liu lab also used natural and engineered...generation of base editors. Compared to BE3, BE4 offers a 2.3 fold decrease in C→G and C→A products as ...yielded an editor with more flexible sequence preferences, evoAPOBEC1-BE4max, which is significantly more...the development of prime editing, which uses a different mechanism than base editing, has allowed scientists...Resources References Chen, L., Zhu, B., Ru, G., Meng, H., Yan, Y., Hong, M., Zhang, D., Luan, C., Zhang,...

... various AAV serotypes can be used to achieve different patterns of expression, researchers often need...empirically for their own experiments. To this end, we offer control AAV in smaller aliquots of 20ul for serotype.... The Addgene viral service has now expanded to offer many of our control plasmids in new serotypes, as...optogenetics lie in the spatiotemporal control offered by the system that allows for specific manipulation...H134R) bound to an mCherry reporter. We now also offer AAVrg for plasmid 83898, which targets expression...Gordon Fishell   Chemogenetics Chemogenetics offers noninvasive methods for manipulating cell activity...dependent version of the DREADD hM3D is now also offered as AAV1 (along with AAV PHP.eB, AAV Retrograde,...

...new features to the tool. Resources and References References Adler, B., Trinidad, M., Bellieny-Rabelo...solve the problem: CasPEDIA, a new resource from Jennifer Doudna’s lab at the Innovative Genomics Institute...CasID, to help facilitate direct comparison of different enzymes. CasPEDIA is meant to be a constantly ...plasmids containing that enzyme, and embedded in the References lists on every page you can find direct links...design, etc.). With this feature, you can compare different Cas enzymes to make sure you are getting the desired...the winner!).   Fig. 3 – Comparison of different Cas enzymes using CasPEDIA. Adapted from Adler...

... 2019), and different combinations of ITR and capsid serotypes vary in their gene transfer efficiency....DNA molecule as the gene that they package. In a transfer plasmid, the transgene is flanked by two ITRs,...and rAAV vectors, modifications can affect gene transfer efficiency and transgene expression (Zhou et al...expression can be altered by using ITR sequences from different AAV serotypes that vary in their RBE and T-shaped...restriction digest methods.    Resources and references More resources on the Addgene blog Viral Vectors...Addgene.org Addgene's AVV guide AAV Viral Preps References  Chen, Y., Hu, S., Lee, W., Walsh, N., Iozza...

... scientific conference, whether you are attending in person or monitoring that conference via social media...Fast Company, opportunities to speak at conferences, and offers for postdoc positions. Check out the podcast...write a second. While working on this post, I was offered and accepted a position to work remotely as an ...American Society for Cell Biology (ASCB) 2018 Conference. I don’t exactly know how I was selected to participate...s worth a read since many of the pointers are different from the other links shared in the post. And the...

...and it’s already looking like it’s making a big difference. We can now look at many more transcription factors...Mendenhall: The system is really quite simple but it offers improvements in efficiency and throughput. In adapting... this for tagging any protein in all kinds of different assays, not just transcription factors. The second...quite small—20 amino acids—so we don’t think it interferes with the function of the protein. Myers: We’ve...Mendenhall: The benefits are two-fold: CRISPR/Cas9 offers high efficiency of inducing double strand breaks... surprising to us that there aren’t a hundred different versions of this out there. Addgene: Why was it... They could also switch from FLAG to GFP or a different epitope. Addgene: Do you have any additional tips...

...delivery of split base editors Article contributed by Jennifer Tsang AAVs are a great gene therapy delivery mechanism...reproducibility as scientists can assemble parts from different labs and experimental systems in their own research...mutants, determined protein expression levels from different promoters, and inhibited gene expression using...contributed by Erin Sanders CreLite, designed by the Eisenhoffer lab, is a new tool that increases temporal and...bringing the two halves of Cre together. Image from Eisenhoffer et al., 2019. This novel tool is practical...organ culture, as well as cell lineage studies. Eisenhoffer and his colleagues evaluated CreLite for cell...eye, muscle and epithelial cells, as well as in different stages of development, ensuring the breadth and...

...Agrobacterium-mediated DNA transfer, each GOI is usually expressed from a different plasmid, which may lead... entry vectors (pUC57-L1L4 and pUC57-L3L2) that offer the advantage of using restriction digest cloning...plasmids to study PPI using Förster Resonance Energy Transfer (FRET, Heckler et al., 2015) or ratiometric Bimolecular...consists of 4 plasmid toolkits generated with 4 different, binary 2in1 vectors with all possible combinations...change in protein conformation in response to light, offers a way to spatially and temporally control cellular... or dark- state of the photoswitchable proteins offers a powerful approach to control protein-protein ...

...al. designed plasmids containing Cas9 and two different gRNAs, and electroporated them with an ssODN repair...experiments in S phase zygotes at 18 hr post fertilization. They observed higher targeting efficiency than...41/42 embryos were non-mosaic at 3 days post fertilization. Since Ma et al. designed a repair template ...about the creation of “designer babies.” The differences in editing between iPS cells and embryos make...off-target effects, but this advantage likely won’t transfer to all genes we’d like to target. In addition,...embryos to only 60%? Unfortunately, given the differences between CRISPR repair in embryos and iPS models...available with current techniques like PGD.  Reference Ma, Hong, et al. "Correction of a pathogenic gene...

... to what’s happening at different points within the wood. There are different types of cellulose fibers...number of DNA binding proteins with Cas9 guided by different promoters. The challenge in building genetic circuits...getting enough regulatory proteins that don’t interfere with each other. CRISPR is so orthogonal and programmable...then you’d need the ability to turn on genes at different times. It requires more complex synthetic networks...circuits, they are extremely orthogonal. It’s a different perspective and you can layer them for more complicated...for multi-input CRISPR/Cas9 genetic circuits References: Nielsen AA, Voigt CA. Multi-input CRISPR/Cas...

...numerous variants for the different types of opsins available and their differences and uses have been discussed... the different variants [1, 2, 4]. And, of course, the best way to learn about the many different opsin...about this in detail later). But what are the different steps in an optogenetics experiment and what are...available, instructions for obtaining a material transfer agreement, and list of vendors (such as the vector... core at the University of North Carolina) that offer his constructs. Importantly, control viruses (not... or follow him on Twitter @derekpsimonphd.    References 1. Tye KM, Deisseroth K. Optogenetic investigation...