...the recombinase handles the installation of the donor sequence. The latest work evolves and engineers ...D3L, releasing the autoinhibition and stimulating the methylation of nearby CpG sites on the target DNA...problematic for low-abundance targets or natively isolated complexes. To address these limitations, the Funabiki...Funabiki Lab developed a technique called Magnetic Isolation and Concentration (MagIC)-cryo-EM that combines...streptavidin-coated magnetic beads through an assembly of biotinylated SpyTag-SpyCatcher linkers and spacer proteins...in sample loss. Ci Ji Lim’s lab found that adding Late Embryogenesis Abundant (LEA) proteins, small proteins...particle orientation distribution and Fourier-shell correlation curve (D), and the reconstructed 3D map with ...

...receptors, or post-translational modifications on those proteins, like glycosylation patterns. Surface ...only be interested in one of the various cell populations; for example, CD8+ T (“T killer”) cells. Typically... simply “dump” them by gating on the negative population.     Figure 2: Dump gating labels all...allowing for a flow analysis to be shifted to a later day. Note that intracellular labeling must be performed...method, differences in proliferation between cell populations can be visualized, achieved through, for example...heterogenicity of proliferative behavior in a cell population can be seen as a stepwise decrease in signal ...easily gated on during the analysis. Conclusion Congratulations! You made it through this blog post and hopefully...

...cleavage promotes collateral cleavage of other RNAs. As with other Cas13s, this collateral cleavage does ...These small, but mighty type VI-D enzymes are the latest tools in the transcriptome engineering toolbox....identified other advantages, including Cas13d-specific regulatory proteins and high targeting efficiency, both ...accessory protein RspWYL1 increases both targeted and collateral RNA degradation by RspCas13d. RspWYL1 also increased...WYL domain-containing proteins may be broader regulators of Cas13d activity. This property makes WYL proteins...counterpart to anti-CRISPR proteins that negatively modulate the activity of Cas enzymes, some of which are...Konermann et al. compared CasRx to multiple RNA regulating methods: small hairpin RNA interference, dCas9...

...Roger Tsien Commemorative Conference Awards. Congratulations to the five winners! Abhi Aggarwal Brandán... a conference of their choice while honoring the late Michael Davidson and Roger Tsien who have played...thiol-containing acceptors. These hydropersulfides modulate protein activity and have important roles during...opioid use disorder. Her goal is to identify cell populations in the brain that contribute to the development...functional contribution to a variety of behaviors related to addiction and pain. Once these are completed...neurons using GCaMP7, measuring calcium signaling related fluorescence during morphine administration and...resulted in mechanical allodynia. With the dual regulation, she could then use pulses of blue light to activate...

...plasmid-based tools for rare disease research. Congratulations to the winners! Below, we highlight their research...patient-mutation bearing knock-in mouse model for CTD. Congratulations the Association for Creatine Deficiencies! ...identify therapeutic targets in the future. Congratulations Mark Mellett! Siddharth Prakash: X chromosome...50-100 times higher than women in the general population,” says Dr. Siddharth Prakash, an associate professor...The lab hypothesized that this is abnormally deregulated in TS and can lead to cardiac developmental defects...cause heart problems in TS,” says Prakash. Congratulations Siddharth Prakash! Terry Hébert: How GNB1 mutations...GPCRs) detect molecules outside of the cell and translate those signals into cellular responses. For example...

...of cells. The antibiotic in your plate might not work. Check the plates! Streak on them an E. coli  strain...help you at this stage and that can be used to simulate in silico cloning. This will definitely help in...enzymes that you are using are not blocked by methylation like XbaI, ClaI, etc. If you are doing multiple...Design primers that overlap at least 24 bp with the template and if the sequence has high GC or AT content,...if you are having difficulties in amplifying a template, particularly from a large genome, you can recheck...have no colonies, check that the antibiotic in the plate correspond to the antibiotic resistance marker present...growing fine, it is probably time to prepare new plates with a fresh stock of antibiotic. Many small colonies...

... as law, medical/technical writing, clinical, regulatory, product development, business development, consulting...foreign workers in specialty occupations. US regulations define a "specialty occupation" as requiring ...paths are not the only place to develop “industry related skills”. Many career paths in academia as well ...look broadly at opportunities to develop skills related to their areas of interest. Additionally, since...institutions of higher education nonprofit entities related to or affiliated with institutions of higher education...applicant works at institutions of higher education, a related or affiliated nonprofit entity, or nonprofit research...awards, media reports, and membership in groups related to the foreign national’s field.” (Matter of Dhanasar...

...wells plates onto agar q-trays where the samples must be spread much farther apart before plating. With..., we can prep two 96 well plates in just under 3 hours or one 96 well plate in about an hour. Automating...Beckman Coulter’s Biomek FXP. We had a previous relationship with Beckman Coulter and that certainly played...too much and dying.  Also, we added a couple of platforms that fit 48 well q- trays allowing us to do up...96 tips for easy sample transfer between 96 well plates. The second pod can pick up 8 tips and position... robot is called the Qiagen BIOROBOT Universal Platform and it suits our plasmid prepping needs perfectly...on a larger scale. Using the BIOROBOT Universal Platform is ideal because it eliminates the potential of...

... real challenge is finding relevant papers. The latest installment of the Early Career Researcher Toolbox...highlight four tools for finding journal articles related to your actual interests while also staying on ...around since 1996, but is getting an update! The latest version can be found here. The new PubMed will ...these types of accounts can help you find papers related to a particular topic or that were published by...tweet about papers too. You can follow journals related to your field of research or larger journals that...can bookmark a tweet in order to easily access it later. Pro tip Checking the number of retweets, likes,...who published the most papers with your keyword populated at the top of this filter. A unique feature of...

...of an outbreak of the virus in Brazil that was correlated with greater rates of infection and rare, but...Zika virus is expected to expand into heavily populated regions of South, Central, and North America. .... The first was a new class of programmable riboregulators, called toehold switches, which allow for the... 2014). The second was an in vitro paper-based platform that allowed gene circuits to be deployed out ... had been working on advancing the paper-based platform toward a more field-ready format when the Zika...our manuscript to the journal Cell just six weeks later. The Zika publication timeline I think we can speak...we say the timeline to develop our paper-based platform into clinically relevant diagnostics for the Zika...

...genotyping, is critical to demonstrating causal relationships between genotype and assayed phenotype. Verifying...and T7E1 have been widely adopted due to their relative simplicity and low cost. The problem with these...editing events that occur in less that 5% of the population (Fu et al. 2013, Vouillot et al. 2015). Meanwhile...consuming and cannot be applied to heterogenous populations (Bell et al. 2014). Further, Sanger sequencing...Low-throughput, inappropriate for heterogenous populations, low sensitivity Targeted Amplicon Sequencing...list of putative off-target sites that they can later sequence for mutations. One method a researcher ... detection rates mean the investigator can be relatively certain that their samples don’t have off-target...

...in 16 wells of a 96-well plate, allowing 6 conditions to be screened per plate. The results indicated that...enzymes that seal breaks in DNA by joining 5 ́-phosphorylated DNA termini to 3 ́-OH DNA termini (1-4). In...interrogation of multiple potential SNP sites (15). The related LCR technique uses two sets of probes to achieve...the reaction. This “peeling back” of the 5 ́-phosphorylated base can be observed in the crystal structures... pools were prepared consisting of one target (template) strand and four upstream probes and four down-stream...strand covering all 16 possible NN pairs in the template. The probes were designed such that each possible...to screen additional ligases, conditions, and formulations and has led to the development of the new HiFi...

...efficiency of Cas9 cleavage is relatively high while the efficiency of HDR is relatively low, meaning that most...without double strand breaks (DSBs) or donor DNA templates. How does this work? First, an engineered prime...reverse transcriptase domain uses the pegRNA to template reverse transcription of the desired edit, directly...increase activity, enhance binding between the template and primer binding site, increase processivity...different genomic sites compared to PE1. The pegRNA: A template and guide all in one The other important component...pegRNA is a guide RNA that also encodes the RT template, which includes the desired edit and homology ...remake that strand using the edited strand as the template. A third prime editing system called PE3 does ...

...optogenetics differ are: Timing Targeted manipulation Controlling stimulation Invasiveness  Choosing the best...Controlling Stimulation Some studies rely on delivering reversible or varied amounts of stimulation to receptors...where drawn out effects are required. Targeted manipulation Both optogenetic and chemogenetic tools can ...as the light source can be easily and quickly manipulated or turned off. In chemogenetics, while different...injected, this is not as precise and the offset of stimulation is gradual. Invasiveness Both optogenetic and...intracranial implant Noninvasive Controlling Stimulation ExogenousEasily controlled EndogenousNot easily...blog posts Learn more about precise targeted manipulation Resources on Addgene.org Browse all viral ...

...medical journal. Decades later and after years of painstaking work trying to isolate the causative gene for...role of the MECP2 gene, and highlights some of the latest breakthrough therapeutic approaches. What is Rett...et al., 1999). We now know that MECP2 binds to methylated CpG dinucleotides and CAC trinucleotides during...& Bird, 2019) and is a global transcriptional regulator of thousands of genes. Studies have suggested ...with the minor groove of AT-rich DNA sequences to regulate transcription (Baker et al., 2013). Mutations ...in animal models, Rett syndrome is a strong translational candidate for gene replacement therapy. In 2014... expand as Rett scientists deposit their MECP2-related plasmid tools with Addgene and share additional...

...investigator generates a cell population where all genes are knocked out at the population level, but each individual...be targeted. Cells failing to upregulate the protein following stimulation would then be separated by sorting...knocked out. In this genetically heterogeneous population, cells behaving differently (e.g. different growth... response to a cytokine known to cause the up-regulation of an important surface protein (e.g. PD-L1 on...were kindly donated by six different research constellations: Doench/Root (2), Zhang (3), Wu (4), Yusa (...The different labs use different algorithms to calculate which would be the best gRNAs. Interestingly, ...your own favorite Reference Library, for example related to strategies beyond SpCas9, or species not currently...

...phosphoproteins Protein phosphorylation is one of the most abundant forms of post-translational modifications in...questions surrounding serine phosphorylation by adding this phosphorylated amino acid to the genetic code...participates in y. Depletion of your protein may upregulate an alternative pathway or induce other compensatory...cloning have been utilized to create recombination template vectors, to perform multiple site mutagenesis ...where signaling dynamics are influenced by the relatively long life-cycle of a protein from expression ...plasmids required to make singly or multiply phosphorylated proteins (B40 OTS and pCRT7-GFP). To demonstrate... either one or two phosphoserine residues cotranslationally inserted in their canonical positions (SP218...

...arabinose making it difficult to regulate quickly. Arabinose also circulates within growth media and can’t...created a bacterial lawn on an agar plate, put a mask over the plate containing the pattern of the image...image, and shined blue light over the plate. Areas on the plate that are blocked by the mask will not produce...May 13, 2021 The original AraC transcriptional regulator Their system is based on the AraC protein that...transcription.However, this system lacks spatio-temporal regulation, and is difficult to reverse, making it unsuitable... show that you have less heterogeneity in the population and you can do all of these more spatially confined...

...worthwhile to isolate individual clones from that population. You can additionally calculate the minimum...used as a preliminary validation on your bulk population. This way you can ensure you aren’t looking for...performed yielding a graph of edits in the input population or clone.   How to perform a TIDE First, ... sequence the PCR product of both an unedited population and your Cas9-targeted cells. Simply upload both...Validating knock-in mutations You’ve introduced a donor template along with your gRNA and Cas9 combo and are hoping...precisely estimate editing frequency in a bulk population and also identify edits in individual clones....reads from your edited sample to an untreated population. Software such as CRISPResso can help with the...

...demand for the related plasmid. Under such circumstances, we would observe this same correlation, but it wouldn...here? Do papers associated with Addgene deposits accumulate more citations than those without Addgene deposits...aggregated this much, you can see the 2013 ‘bump’ related to the CRISPR/Cas papers and Addgene deposits, ...Addgene, in contrast to other reasons for the correlation. However, determining this exactly is difficult... of publication. Table 1 provides an example calculation of the weights. Table 1: Journal Weighting Example... remaining difference caused by depositing the related plasmids with Addgene? Probably not all of it. ...still be controlled for. As an example, plasmid-related areas of science could be growing more quickly ...