...strategies Publication System nickname Photoactivatable moiety Cas9 variatn CRISPR protein application Split...improvements to CRISPR technology since its initial applications for genome engineering in 2012.  Many of these...temporal and spatial control. Optogenetics uses genetically encoded tools, such as microbial opsins, to control...enzymatic activity. Photoactivatable genome modifications by NHEJ and HDR using split systems Later in...mutations via NHEJ (frequency of 20.5%) and induced modifications by HDR (frequency of 7.2%). The authors additionally...C714, generating paCas9-2. This change did not significantly alter the system’s efficiency at generating ...particular interest since blue light has limited applications in vivo. UV and blue light is readily absorbed...

... do anything. Viruses are complicated and they perform complex biological tasks, both of which are factors...C virus RNA quantification as measured with branched-DNA technology." Journal of clinical microbiology...factors in the technical challenges they can present. Many researchers find themselves plagued by troubleshooting...hail Marys that have helped me troubleshoot some technical obstacles when working with virus: 1. Viral vectors...vector may undergo genomic rearrangements during replication. To avoid genomic rearrangements, try amplifying...Different titering methods can reveal differences in physical versus functional titer. If you have low titers...cells, replace the medium with half the volume typically used (e.g., use 5 mL medium on a 10 cm plate)....

...the OpenWorm website. microPublication microPublication is a unique publication for the worm community...into larger publications and would otherwise be lost in lab notebooks forever. microPublications are peer-reviewed...aims and approach of CeNGEN, check out their publication.   3’UTRome The 3’UTRs of mRNA are important ...Biodesign institute developed a 3’UTR database specifically for C. elegans. This database provides detailed...normally one figure and can be negative and/or even replicated studies to show reproducible results. This a ...referenced. In addition the data published in microPublications is curated and added to other databases including...including wormbase and flybase. microPublication is run and maintained by members of the worm community...

...experiments. However, the technical demands of using these wireless devices are significantly higher than standard...optic cable is threaded through—and fixed to—an optical insulator called a ferrule (below). The fiber optic...portion of optogenetics (though it does require a significant amount of equipment, most of which is relatively...There are several important considerations with fabrication of the fiber optics needed for optogenetics. ...point; the fiber optics need to be polished to significant smoothness to prevent drastic loss of efficiency...maintained during the entire protocol, otherwise optical stimulation will be erratic, opsin activation/inactivation...stimulation, the rate of movement of the animal significantly increases. Before you get your experiment started...

...May 22, 2019 Smart Servier Medical Art This site has 3,000 free medical images organized into four main...software that let me create graphics that better communicated the science in my blog posts. This post highlights...free tools which will hopefully also help you communicate your science, whether it’s in presentations, ...number of drawing tools, so it’s harder to draw intricate figures, like a brain or a mouse. Vectr Vectr ... over 20,000 icons is designed by professional medical illustrators with input from life scientists. It...without the watermark, which are better suited for publications, if you sign up for a paid account.  I’m proud... anatomy and the human body, cellular biology, medical specialities, and general items. Individual images...

...origin of replication (which controls the host range and copy number of the plasmid) and typically include...Description Origin of Replication (ORI) DNA sequence which allows initiation of replication within a plasmid...point for PCR amplification or sequencing. Primers can be exploited for sequence verification of plasmids...plasmids are small circular pieces of DNA that replicate independently from the host's chromosomal DNA....Minimally, lab-created plasmids have an origin of replication, selection marker, and cloning site. The ease... plasmids and the ability of plasmids to self-replicate within a cell make them attractive tools for the...plasmid by recruiting replication machinery proteins Antibiotic Resistance Gene Allows for selection of plasmid-containing...

...   Figure 3: Each publication featured in BioGRID ORCS has a dedicated summary page with PubMed...result interpretation. The analytical method for score assignment and significance thresholds used by the ...favorite gene was described in a CRISPR screen publication. You see this mentioned in the results section...new freely available resource developed by the Biological General Repository for Interaction Datasets (... to allow facile queries of published data by biomedical researchers and ready access to stably archived...manually curated and annotated from more than 45 publications, beginning with the seminal research articles...CRISPR screen results available to the entire biomedical research community, but also allows cross-interrogation...

...their undomesticated counterparts) - not exactly species that jump to mind when we think “biomedical research...ways these animals have impacted humans through biomedical research. The Camelid family is composed of ...research". The family entered the biomedical field’s spotlight after researchers discovered heavy-chain...to sdAbs becoming valuable tools for diverse applications. For example, RANbodies can be used as an alternative...chromobodies,” which are single-domain antibodies genetically fused to fluorescent proteins and used to visualize...therapeutic was approved in 2018, with many others in clinical trials (Jin, et al. 2023). In addition to therapeutics... excitement around using sdAbs in diagnostic applications. For example, fluorescently and radioactively...

...Rep78/68 play a key role in AAV genome replication. Replication initiates from the AAV genome ITRs on ...Srivastava A (2019) Capsid Modifications for Targeting and Improving the Efficacy of AAV Vectors. Molecular...contains the proteins needed for the virus to replicate,  and the transfer plasmid which contains the ...aka the RepCap plasmid encodes two genes: the replication (rep) gene and the capsid (cap) gene. In wild-type...its products. Created with BioRender.com   Replication (rep) gene The rep gene encodes four proteins...proteins that are required for viral genome replication and packaging: Rep78, Rep68, Rep52, and Rep40. Rep gene... domains, all four Rep proteins share a common helicase and ATPase domains. To function, the Rep proteins...

... tactics of prokaryotes to deliver the technological biological blockbuster known as the CRISPR (clustered...genes, which code for polymerases, nucleases, and helicases, were also disrupted to elucidate their role in...destroy the phage upon re-exposure (2, 3). More specifically, specialized Cas proteins snip the foreign DNA...distinct types and subtypes using polythetic classification based on evolutionary similarity (5). Type ...2013 (7, 8). Nine months later, more than 1500 publications describe work to improve the tool’s specificity...various species and describe an assortment of applications. Despite the ubiquity of CRISPR/Cas toolkit,...programmable platform well-suited for various innovative biological and translative technologies. Addgene has empowered...

...lab while the publication page contains all the plasmids deposited from the same publication. Next up, we... Addgene’s plasmids is an indication of popularity. A yellow flame indicates that a plasmid has been ordered...through the depositing lab page, then onto the publication page, and finally on down to the individual plasmid...Great! Now, if I want to use this plasmid in a publication, how do I know who to acknowledge? Depositing...plasmids that are available through Addgene. Publication: If the plasmid has been published, the paper...the bottom of the page that help you cite this publication in your own work: How to cite this plasmid: The...depositing lab whenever their plasmids are used in a publication. Addgene makes this easy by providing a template...

...methods. Since molecular, ecological, and reproductive confinement typically require very little effort...Development Fellow at the Wyss Institute and Harvard Medical School. Scientists making transgenic organisms ...the entire wild population with unpredictable ecological effects. Because the consequences of such a mistake... to implement and have been tested in yeast5. Ecological confinement involves performing experiments in...experiments attempting to build gene drives in tropical mosquito vectors of diseases such as malaria and...they should not be relied on exclusively because historical studies of pathogen research have conclusively...strategies is strongly advised. The effects are multiplicative, and there are very few gene drive experiments...

...Castano Corsino, a junior faculty member at Harvard Medical School/Brigham and Women’s Hospital (BWH). Based...like IMP, bring closer than ever (although not physically) the ideas and experience from talented professionals... IMP USA-Europe not only provide the specific technical and logistic support to increase the chances of...professional opportunities that are outside of our typical boundaries, and will arrive to Spain at one point...international mentors. Universities from five different geographical locations in Spain were enrolled: the University...represented a diverse group of individuals with technical and career path needs, and came from the universities...that their knowledge and skills had improved significantly when compared to their pre-program self-assesment...

... editing in Escherichia coli. Acta Biochimica et Biophysica Sinica. https://doi.org/10.1093/abbs/gmab036...for successful modifications, efficiency can be low, especially for larger modifications. What’s the solution...tools available for bacteria and some of the applications for which they’ve been used. The beginnings ... of Enterobacteriaceae, without the need for modification. This finding suggests that the system may be...eukaryotes (Qi et al., 2013). As in other systems, catalytically dead (dCas9) targeted to a promoter or gene ...gene body can repress transcription by physically blocking the elongation complex from binding the DNA or...responsive promoter to drive expression of a catalytically dead Cas9 enzyme allows reversible repression...

...where you assume your technical skills define you and that the other not-so-technical skills you’ve built...my job. The same can be said about teamwork, communication, writing, management, and creativity; I have... individual with more going for you than your technical expertise in a specific field. While searching...built over time are only applicable to those positions you have already held. Imagine this situation: You...have the desired degree, you have mastered the applications they use, you’ve gained experience working with...under your belt from your last job. You knock the technical portion of the interview out of the park. It’s...hiring team will also want to examine not just technical skills, but also transferable skills, before offering...

...mismatches on modification by AsCpf1 (red) or LbCpf1 (blue). Mismatch positions are indicated below each ... this publication at Addgene. Makarova, Kira S., et al. "An updated evolutionary classification of CRISPR-Cas...it lacks Cas9’s other HNH endonuclease domain, indicating that Cpf1 functions differently. Since Cpf1 loci... Direct synthesis of these crRNAs should be significantly cheaper than that of the ~100 nt crRNA/tracrRNA... off-target editing rates, determined using sophisticated genome-wide analysis. At many of its computationally...compared the ratio of total off-target to on-target modification for AsCpf1 and LbCpf1, and found that both orthologs...-target editing in a cell culture model. The application of Cpf1 to genome editing is exciting both in...

...Cell-Type-Specific Gene Editing." Journal of the American Chemical Society 140.21 (2018): 6596-6603. PubMed PMID...valuable to scientists, there are some potential complications that must be considered when using this method...alternative approach, which avoids many of these complications, is to directly deliver a ribonucleoprotein ...used for most of the current genome engineering applications of CRISPR including: generating single or multi-gene...pathways. This makes Cas9 RNPs useful for CRISPR applications where limited expression of Cas9 is required... of eventually using this method for in vivo applications. This technique utilizes Cas9 proteins harboring...Liang et al. 2017).  PEG-mediated methods, while technically less difficult to perform, often show low efficiency...

...require grant seekers to use online applications with significant character limits for the written portion...development, certification, degrees, foundations, coaching and even Twitter Chats dedicated to the Grant...the candidate application process, the candidate’s ability to write a grant application through a written... much more than writing. In fact, many grant applications and grant makers have moved away from long written...portion of the grant application. They have increased their demand for stronger budgets with clear budget...duties can range from coordinating the grant application and submitting it, to evaluating the capacity...especially in the sciences and by extension to scientifically oriented nonprofits. Like Deb, I worked for...

...dynamic, physiological processes. Re-engineering GFP to increase its range of color and application Almost...through mutagenesis in order to improve its physical and biochemical properties. In 1995, Roger Y. Tsien described...transgenic mice. Purification: GFP can be used as a general epitope tag for protein purification and a number...scientists to begin to study GFP and deduce its biochemical properties. Now GFP and its fluorescent derivatives...GFP is used in research across a vast array of biological disciplines and scientists employ GFP for a wide...their experimental needs, and some of the common applications in the lab. Why Green Fluorescent Protein?...others) Neutral mutations    A multitude of applications Due to its size and ease of use, GFP and other...

...after subtracting out non-specific binding). Historically, glutathione-S-transferase (GST) fusion proteins...advantage of a fusion protein is the efficient purification of an already immobilized target protein. The...immobilization method. One possibility is covalent modification of the purified target protein with the vitamin...derivatives streptavidin and neutravidin) is for all practical purposes covalent. Therefore, protein immobilization... four biotin-binding sites per molecule so, theoretically, saturated avidin could form tetramers of biotinylated...well-controlled. The lack of site selectivity typically diminishes the apparent affinity for the ligand...However, large amounts of the target protein are typically required due to the relatively low efficiency ...

...genomic modifications, is very robust in C. elegans, and the methods for HDR-mediated modification continue...permission from the Genetics Society of America. Applications of this system Dickinson et al. have developed...Used with permission from the Genetics Society of America. To use this SEC-based system, first design...use and highly efficient for C. elegans genome modification. Figure 2: Schematic from Dickinson et...system could be used to make many other genome modifications, including targeted mutations. SEC-based systems... in cell culture. In addition to CRISPR/Cas9 applications, SEC selection could replace other, more restrictive... of print] PubMed. Find plasmids from this publication at Addgene. Other strategies for CRISPR/Cas9...

...mammalian cells often relied on chemical (CaCl2, cationic liposome) or electrical (electric current forming ...research since their first use in the early 90’s to genetically modify primary cells. Amongst the different vectors...particles and the virus particles produced could not replicate. However, Mother Nature found a way to re-create...packaging plasmid forming a vector that could create replication competent virus. Even though this system was ...its safety was a disadvantage particularly for clinical development. In addition, MoMLV is inefficient...still a potential risk for the generation of replication competent lentiviruses (RCL) especially if you...factors in HIV positive cells could make the virus replication competent. The 3rd generation of lentiviral...

...post was updated on Jul 27, 2020. CRISPR, and specifically Cas9 from S. pyogenes (SpCas9), is truly an ...use, functional in most species, and has many applications. That said, SpCas9 is not the only game in town... makes this molecule unique, and the various applications of Cas13a. The origins of Cas13a: An RNA cleaving...specified by the crRNA sequence, it adopts an enzymatically “active” state rather than reverting to an inactive...discussed below. Subscribe to CRISPR Blog Posts Applications of Cas13a   Figure 1: Using Cas13a as...) is amplified using Recombinase Polymerase Amplification (RPA) or Reverse-transcription RPA (RT-RPA),...fluorophore. The fluorescent signal can be used as an indicator to determine whether the target sequence is present...

...tropism, replication kinetics, and cell damage profiling of SARS-CoV-2 and SARS-CoV with implications for ... from their original sources. Figure 1: Biological repositories that distribute mouse strains, plasmids... plasmids, cell lines, and much more have been critical for pandemic research. Image from Reproducibility...this virus. One such example is cell lines. The American Type Culture Collection (ATCC) is a private, nonprofit...Cell Bank, part of the National Institutes of Biomedical Innovation, Health, and Nutrition in Japan. These... Another example of reagents that have broad applications but have proven useful to the study of SARS-...vectors VSV-G and psPAX2, which were used in a publication by Xiuyuan Ou, et al. that characterized the ...

...overall. Communication is the issue Much of the problem can be attributed to communication, and in particular...particular communication about experimental methods and approaches. It’s very easy to think that the methods...that they were doing something differently. Communication comes in many forms—from transparency about ...them troubleshoot and understand. All of this communication and sharing fits together to make for a transparent...for pushing for more transparency in science communication falls on the shoulders of journals and publishers...can feel like yet another hurdle in front of publication when all they really want to do is get the paper...we take our responsibility to push for clear communication of science very seriously. That’s why at Cell...

...skills The ability to think critically and carry out in-depth research is applicable to a wide range of industries...many opportunities available to those who are scientifically minded! Listen to Biotech Founder Michael Koeris...Scientific Advisor Lab Manager Patent Lawyer Technical Writer Meteorologist Journal Editor Textile ...organised, reliable, and can work independently. Practical outdoor skills are needed in a variety of roles...Breeder Scientific Illustrator Plant Breeder Zoological Worker Conservationist Documentary Director ...range of roles, including: Science Reporter Technical Writer Statistician Bioinformatician Science...Marketer Software Developer Learn Science Communication Skill from a Student Group Like Science in the...

...cells and statistically analyze cell movements. By using cells labelled by fluorescent chemicals or by other... steady state or under physio-pathological challenge. However technical specificities have to be taken... required for a growing organism to make the complicated transition from a clump of cells to a fully differentiated...mobility than the static microscopy images of chemically coloured lymphoid organs did in the past. How...fluorescent protein or dye. It separates the cells mechanically in a vibrating nozzle, imparting a positive ... millimeter in depth. Two low-energy photons (typically from the same laser) cooperate to cause a higher-energy...reporter constructs in the immune lineage. By specifically labeling T cells, B cells, and antigen presenting...

...target antigen as canonical antibodies. They also have the advantage of being significantly smaller, meaning...binding to cell surface receptors. These applications are typically used for investigating signaling pathways...tool that can be used for a variety of research applications. Within the immune system, antibodies are produced...antibodies to bind proteins is useful for research applications as well because they allow scientists to target...Epitopes are between 5-8 amino acids long, with a typical length of 5 or 6 amino acids (Cruse et al., 2004...recognize epitopes that full antibodies would not physically be able to reach. While a full antibody is ~150...150 kDa, a nanobody is 12-15 kDa.   Research applications for antibodies You can use antibodies for experiments...

..., the lessons learned in pursuing one technological application can benefit many others and accelerate...vitro, and what obstacles must be overcome for clinical success. ART and HIV-1 reservoirs HIV-1 infects...toxic proteins, CRISPR/Cas9 SAM caused apoptosis, indicating a true “shock and kill” response. Cutting HIV...create mutations in genes essential to HIV-1 replication. However, this approach could permit some viral...along with Cas9 in the T-cell line 2D10. PCR amplification and Sanger sequencing of pooled samples showed...-1 strains, Cas9/gRNA lentiviral expression significantly reduced the HIV-1 copy number, although the ...these methods represent exciting advances in preclinical HIV-1 research that should be pursued in animal...

...selection and directed evolution." Journal of the American Chemical Society 136.46 (2014): 16299-16308. PubMed...di-GMP and cyclic AMP-GMP." Journal of the American Chemical Society 135.13 (2013): 4906-4909. PubMed PMID...aptamer soup by exploring the development and applications of fluorescent RNA aptamers in Addgene’s collection...Broccoli, and useful for quantitative studies. Specifically, Corn was developed to quantify RNA transcription...biotin (the addition of biotin allows for the purification of specific RNAs from cells using this tag, ...TO1-biotin and TO3-biotin contain an acetate modification that reduces nonspecific binding with RNA by...non-coding RNAs in mammalian cells." Nature communications 9.1 (2018): 656. PubMed PMID: 29440634. PubMed...