...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...

...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...

...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...

...colleges, private universities, and medical schools. I will emphatically deny that I have a magic formula..." manual on applying for faculty positions in biomedical science. My motivation was to share my experiences... do need multiple first/corresponding author publications in quality journals. It helps to come from the...a job. The currency of scientific success is publications and grants. 2. Write succinct and easy to understand...told all of the nuances of the research in as technical of language as possible. Seriously. My suggested...humbling. My conviction that I had a flair for communication was rapidly dispelled. She would ask me, "Is... trying to say X." She would then look at me quizzically and ask why I didn't just say that? Bottom line...

...recombinase polymerase amplification (RPA) using a commercially available amplification kit. In this case,... RT-qPCR tests to diagnose COVID-19 and some serological tests to determine past exposure. However, the... RNA is extracted and subject to isothermal amplification by primers that target the E and the N2 gene...reverse transcriptase to produce cDNA. Isothermal amplification is done at one constant temperature, eliminating...rapid detection.  Highlights: Cas13a is not catalytically active in the presence of two or more mismatches...that exhibit fluorescence upon cleavage. Upon amplification by the RPA primers, the Cas12a-crRNA binds to... SARS-CoV-2 detection, AIOD-CRISPR without preamplification was able to detect as low as 1.2 copies DNA...

...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...

...established applications of the original hybridoma monoclonals, we anticipate that our chemically identical...kidney development. Despite their ubiquity and significance, these receptors have proved challenging to ...specific ligand-binding functions, all key to basic biomedical research and drug development. Integrin structure...long legs that protrude from the cell membrane, physically connecting the extracellular environment and ...signal cascades, and allowing agile responses to mechanical forces in the cell’s microenvironment. The actin...and (C) extended-open. When a ligand binds, it typically shifts the integrin from its closed to its open...resulting in cells that elongated and morphed asymmetrically (Jo et al., 2022). By contrast, LVDP-binding...

..., 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...

...simulate physiological gene expression regimes that cannot be easily achieved with classical gene overexpression...changes in expression of TRIM21-associated genes, indicating that TRIM21 overexpression does not aberrantly...light microscopy, comes from his recent Nature Communications article “SRpHi ratiometric pH biosensors for... super-resolution microscopy.” Endosomes are historically difficult to study in living cells using conventional...250 nm), and studying endocytosis is further complicated by the reliance on pH-based biosensors used to...the Hell lab designed their super-resolution pH indicators (SRpHi) biosensors to meet all of them. These...SRpHi Biosensors here. Richardson et al. Nat Communications. 2017. PubMed PMID: 28924139. FIRE-Cas9 ...

...detected with tyramide signal amplification. Tyramide signal amplification occurs when HRP converts a fluorescently...instead of via recombinant DNA, they can’t be genetically tagged and instead are conjugated to reporters...reporters (i.e. fluorescent dyes, HRP, etc. ) after purification. Antibodies are difficult for an individual ...culture media. Their His tags allow for further purification, if desired, but it's not required for their...some reporters are better suited for particular applications. Read on to learn more about each reporter or...labeled tyramide molecule into a highly reactive radical, which is then covalently linked to residues within...however, compatible with most multi-labeling immunohistochemical protocols where either a directly labeled...

...previous two posts in this series described a practical approach to selecting a bright fluorescent protein...aggregates. This property can be tolerated in some applications (e.g. labeling of cells or tracking promotor...promotor activity), but it is problematic in applications in which the fluorescent protein is used as an inert...During the experiment, the solution is diluted, complicating the interpretation. Because of this, these methods...ultracentrifugation. Sedimentation equilibrium analytical ultracentrifugation yields an affinity and has...most likely in an antiparallel configuration), typical structures known as Organized Smooth Endoplasmic...Constantini et al, 2012). The localization and biological properties of new fusions can be compared with...

...the 4D nucleome. Visualizing specific loci has historically been difficult, as techniques like fluorescent... every genomic locus, and to do so in vivo. Catalytically dead Cas9 (dCas9) cannot induce gene editing... The technique successfully resolved loci with physical map distances of 2 and 75 Mbp, with the calculated...distances correlating with the previously established physical map. In comparing pairs of targets ~2 Mbp apart...the respective hairpin binding domain. Pairs of identical hairpin sequences specify the primary colors blue... colors and a white composite. gRNAs with two identical hairpin binding domains produce blue, green, or...this rainbow of colors, Ma et al. performed sophisticated tracking of multiple chromosomal loci in live...

...would need to be much, much lower before clinical applications could be considered. It is important to ...answer questions of efficiency and applicability. Given moral and ethical concerns surrounding such research...experiments are needed before we can consider clinical applications for germline editing. Although this paper...thus allowing repair to take place before DNA replication. Indeed, they observed that 41/42 embryos were... embryos carried indels at the mutation site, indicating that repair proceeded through NHEJ rather than...attributable to CRISPR editing. Conclusions & implications One striking feature of this paper is the failure...consider that already-tested CRISPR alternatives are applicable in many situations. In the case of hypertrophic...

...in their ability to be specifically and potently activated by pharmacologically selective effector molecules...chemogenetics for research and potential clinical applications. Science 6436: eaav5282. https://science.sciencemag.org...wiring of the brain and the underpinnings of neurological disease, neuroscientists need to be able to ...What is chemogenetics? Chemogenetics relies on genetically introducing engineered receptors that are only...et al., 2019). Using PSAMs PSAMs PSAMs, or pharmacologically selective actuator modules, were developed... such as varenicline, are already approved for clinical use. Varenicline has been further modified to ... create ultrapotent PSEMS (uPSEMs) that have drastically increased potency. Download our chemogenetics...

...is complex Plasmid classification has shifted over the years, and new classifications are still being created... Characteristics and Potential Applications in Biotechnology. Critical Reviews in Biotechnology, 24(4)...us! A plasmid is a small piece of DNA that can replicate independently from its host’s chromosome. Although...genes between bacteria can also have serious implications for public health. Bacteriocin plasmids: microbial...carrying genes for bacterial toxins like colicin typically also carry the necessary resistance genes to help...cells survive their own secretions. Pro tip! Historically, colicin-producing plasmids have been referred...which strains of E. coli contain this F plasmid is critical for successful Gateway cloning. In addition, scientists...

...mismatches on modification by AsCpf1 (red) or LbCpf1 (blue). Mismatch positions are indicated below each ...remove the PAM sequence. Many research and clinical applications require HDR over NHEJ, so comparing HDR ...eclipse any of the CRISPR nucleases in AAV-based applications. Get More Cpf1 Background Here THE ORIGINAL ... 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...PubMed PMID: 27272384 Find plasmids from this publication at Addgene. 2. Kleinstiver, Benjamin P., et ...PubMed PMID: 27347757. Find plasmids from this publication at Addgene. 3. Zetsche B, Gootenberg JS, Abudayyeh...

...science, dating back several hundred years, with historical ties to psychology and philosophy (the mind-body...there are two main decisions to make: 1. Which modification? and 2. Which viral vector should be used? Modifying...chemogenetic tools are designed channels that are chemically activated. Both are introduced in neurons and...excitation or inhibition upon light (or drug) application.  Genetic manipulation can also reduce expression...recombinase on transgenic flox animals to introduce modifications at the DNA level in a temporally and locally... brain area, avoiding side effects from such modification in other areas. An alternative strategy is to...in a certain type of behavior (for a review see Stoica et al., 2013). Vector delivery may also be achieved...