... 49,000 times! Their long-lasting popularity illustrates the utility these tools have in many different...pyogenes with an invariant sgRNA scaffold and cloning sites for inserting the guide sequence (pSpCas9(BB)). ...notice the “V2.0” at the end of this plasmid which indicates that there was a prior version of this plasmid...retroviral). Fun fact: published in 2003, this plasmid predates Addgene’s founding. Find pCMV-VSV-G. pRSV-Rev...

...fantastic K12 education initiatives like BiteScis (https://bitescis.org/) and amazing graduate student writing...writing collaboratives like Oceanbites (https://oceanbites.org/) have already grown out of our workshops...

...These small, but mighty type VI-D enzymes are the latest tools in the transcriptome engineering toolbox....sequence! In bacteria, Cas13d-mediated cleavage promotes collateral cleavage of other RNAs. As with other...efficiency varies widely across guide RNAs, each guide tested with CasRx yielded >80% knockdown. It seems that...maintains its RNA-binding properties, Konermann et al. tested its ability to manipulate RNA species through exon...

...or PolyPhen to approach this challenge, using attributes of cancer variants such as evolutionary conservation...have been two huge challenges preventing us from testing this hypothesis: There was no large scale, broadly...whether gene-agnostic bioassays could accurately test for molecular signatures of variant function and...the second major challenge, we developed assays to test large numbers of cancer variants across a large ...

... want to buy, it is worthwhile to test-drive a couple of candidates. Before you buy a new microscope, ...summarizes properties of fluorescent proteins on websites. To make the best choice, the fluorescent protein...than theoretical brightness for selecting the brightest fluorescent protein. Theoretical brightness Two...

...finishes repair of most types of breaks in tens of minutes – an order of magnitude faster than HDR. NHEJ is...paired end complex The paired end complex then ligates compatible DNA ends together, thus repairing the...Aprataxin, Tdp2) that function to make ends better substrates for ligation. Although we do not describe these...vastly reduces breaks and mutations at off-target sites. Christina Mork contributed to the update of this...

...relevant to the disease in question – cardiomyocytes, adipocytes or neurons, for instance – to see how...Cell Stem Cell paper, for example, we generated hepatocytes to examine the effect of a gene on cholesterol...Musunuru: The CRISPR/Cas9 system is easier to use as testified by the fact that the TALEN kit includes 834 plasmids...

.... ANOVA, t-test, multiple regression analysis, and non-parametric analogous Wilcoxon tests have all been...variation into your data. Try different types of pipettes and pipetting methods to find what’s most consistent...plate. Compare the CT values within each set of triplicates to understand variation due to technical error...reproducibility’s sake!   Fig. 2: Setting up triplicates in multiple configurations can help you identify...

...your search and sorting the results (relevance, dates etc). The links for the titles can generally take...you provide, Google Scholar will send you daily updates with relevant literature. Organizing the literature...margins. If you prefer, printing and hand-writing notes is still possible, but not feasible for every paper... University. She is a fan of photosynthesis and writes about autotrophs and biochemistry on her blog. ...

...splitting removes any exogenous sequences and regenerates a native peptide bond at the split site. ...to that of a single AAV expressing GFP. The lab tested the base editors in vivo and achieved therapeutically...used in human gene therapy trials. They further tested the base editors by correcting a mutation that ...synthetic biologists to use Type IIS restriction sites to easily assemble multiple DNA fragments. This ...

...these plates now” to a fellow team member. But, at Addgene, we use seven different types of “plates” (Fig... meetings. A lot of our LIMS planning involved “plates,” and our scientists and lab technicians always...information Prescribing an official name to all of our plates, tubes, and racks meant we had fewer errors in ...

...allowing an influx of ions into the neuron which generates an action potential (Figure 1). This light-induced...channelrhodopsins to the brain. This delivery route concentrates lots of channelrhodopsins in one region of the...broad transduction of the nervous system which eliminates the need for intracranial injections. When packaged...dopamine is an important neurotransmitter that promotes reward-motivated behavior, if the channelrhodopsins...

...the accumulation of toxic intermediates or the shuttling of the intermediates into other pathways. In cases...of plasmids, the stabilized promoters were also tested in different plasmid backbones that varied in size...gene copy number is affected differently by growth rates when a gene is expressed from a plasmid as opposed...

...DNA-protein interactions or to analyze chromatin states (CHIP-seq, FAIRE-seq, Cut & Run) and to detect ...signal at the same time point. E. coli’s BirA* was tested at its optimum 18 hour time point, but one minute...physiological expression levels; however, knock-in of BoxB sites into endogenous DNA sequences could allow one to...Human genetic disease. RaPID can also be used to test how RNA variants associated with human disease affect...

...protospacer. The resulting double strand break (DSB) stimulates DNA repair mechanisms that can be exploited to...rules that result in a decrease in available target sites. Regulating gene expression with truncated gRNAs...Altering the length of gRNAs targeting different sites of interest thus represents a straightforward means...transcriptional activation or repression at the respective sites.   Many thanks to our guest bloggers Alissa Lance-Byrne...

...then eluted using a high imidazole buffer that outcompetes the interaction between the protein of interest...empirically determined. In addition, some metal ion substrates, such as those containing Co2+, have a lower ...affinity for polyhistidine than more commonly used substrates such as Ni2+ and are therefore more selective... systems, consider using these more selective substrates along with stringent wash conditions.   Find ...

...those sites can be excised by intentional introduction of the Cre recombinase. This is a time-tested system... yet permanent gene excision. Essentially, LoxP sites are knocked in flanking a gene of interest (or a...system but does require knock-in of the LoxP sites, which is more difficult than a simple knock out. Looking...

... form a clean DNA break, Cas3 locates its target sequence and initiates single stranded DNA degradation...exist with unique cutting features and evolved PAM sites, making Cas9 an adaptable choice for many genome...Cas12’s primary difference from Cas9 is that it generates staggered breaks, also known as sticky ends. Cas12a...

...repeat region on its 3’ end. This repeat region associates with the trans-activating crRNA (tracrRNA) which...at the same site, is 2’-fluoro (2’-F), which substitutes a fluorine for the hydroxyl group at the 2’ position...coupling several of them together, for example phosphorothioates with 2’-O-methyl modifications, proved more...Addgene’s validated gRNA repository to find pre-tested gRNA sequences for your next CRISPR experiment....

...science and engineering. “Fix the Institutions” promotes gender equality in careers through structural ...“Fix the Knowledge” or “gendered innovations” stimulates excellence in science and technology by integrating...combinations of donor/recipient interaction should be tested before being ruled out; there are many factors ...the importance of analyzing sex, the National Institutes of Health (NIH) now requires that sex be included...