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... understanding of CRISPR, and perhaps provide insights and ideas for your own research! Classification... abundant of the two, comprising about 90% of identified CRISPR-Cas in bacteria and nearly 100% in archaea...transposases, by leaving out Cas3. Type I is further subdivided into subtypes A–G. These subtypes have similar...Schematic of a Cascade complex (blue subunits) with a guide RNA (green and orange) bound to the target DNA with...with BioRender.com.   Type III Type III is considered the most complex of the CRISPR types and is thought...et al., 2021). IV-A and IV-B lack what you may consider to be an integral component of CRISPR — a nuclease...engineered Cas enzyme, with adaptations for increased fidelity, activation/repression, high precision edits, ...

...UnLOCKING Cas13 (C2c2) was first identified in 2016 by the Zhang lab as an RNA guided RNase (Abudayyeh et al 2016...-related single nucleotide polymorphisms (SNPs) from human saliva. SHERLOCKv2 provides a method to detect... expensive, time-consuming, and complex to use outside of standard laboratories. In the case of the COVID... 2016). Cas13 can be guided by a single CRISPR RNA (crRNA) to cleave ssRNA or mRNA. It also exhibits a...based on DETECTR technology with the mission to “provide a CRISPR-based platform on which an infinite number...2016) C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science 353:aaf5573... Koonin EV, Zhang F (2015) Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell...

...searched for this unique sequence of DNA and both identified lymphocyte antigen 6 complex (LY6A) as a receptor...library of AAV9 mutants that had a small randomized peptide loop inserted in the AAV9 capsid. This mutant library...then screened in vivo in C57BL/6J mice strains to identify variants that reached the CNS following IV injection...hematopoietic stem cells. Its ligand(s) has not been identified. While LY6A or other Ly6 gene family members ...susceptibility. The Deverman and Wilson Labs independently identified LY6A as a receptor for PHP.B by looking for a...total, they tested 13 strains, with seven strains identified as permissive and six as non-permissive to PHP.B...the PHP.B permissive phenotype of mouse strains beside C57BL/6J and BALB/cJ, and the effects of overexpressed...

...Any ∼20 nucleotides of DNA can serve as the target for Cas9’s single guide RNA (sgRNA), provided it meets...unique experiment. Here, we will introduce you to the wide array of Cas9s (many available through Addgene’s...Specificity concerns? If off-target effects are a worry, consider a dual-sgRNA system. This system uses a “nicking...little homology to other regions of the genome and consider using a Cas9 variant with higher specificity (...residue of a protein or knocking out a very small peptide. In these cases, you have little flexibility as...specific? Perhaps you need to introduce a single nucleotide modification at a defined amino acid to mimic...requirements, giving it more flexibility. A consideration with this tool is that the desired edit will...

...range, identify a few of the features that confer broad host range in plasmids in nature, and provide tips... meliloti, Rhodobacter sphaeroides, R. capsulatus, Rhodopseudomonas spheroides, Salmonella spp., Serratia...equally. Only a handful of plasmids have been identified as broad host range plasmids. The properties ...are not fully understood, but scientists have identified some of the features that confer broad host range...metabolic load (Meyer 2009). Figure 1: Idealized examples of a narrow host range plasmid with only...Porphyromonas gingivalis, Proteus mirabilis, Providencia spp., Pseudomonas spp., Rhizobium leguminosarum... M. xanthus, Proteus rettgeri, P. mirabilis, Providencia stuartii, Pseudomonas spp., Rhizobium leguminosarum...

...accurately communicating ideas in a content-rich, easy-to-understand set of PowerPoint slides. What are some of...learned as a healthcare consultant and give you an idea of what a career in this field involves, and why...a defined path forward. Communication is key as ideas must be relayed quickly both internally and externally..., where case managers are encouraged to use the wider firm’s resources when their teams are facing a crunch...approach brings to patients, doctors, and insurance providers, and is crucial to how our clients’ products will...you’ll have the opportunity to learn the business side of consulting cases, while feeling like you are ...to get your foot in the door When I was first considering preparing for and transitioning to a career in...

... let’s say you identified gene target X for BRCA-deficient breast cancer and identified several candidate... of two approaches. The first route involves identifying a gene/pathway to develop a drug for (aka a druggable...to their target, or their direct target can be identified if it isn’t known. If a strong candidate emerges...put into them in those later stages. Target identification Not all diseases are fully characterized or...simple pathology. In these cases, preliminary identification of potential targets by CRISPR can accelerate... activate, or knockout many genes at a time to identify ‘hits’ - genes that affect disease progression...off target effects, both of which make it a non-ideal drug. If a drug selectively kills the wildtype line...

...need to be inside of a gene’s open reading frame, in fact, they can be activated a considerable distance... gene expression are all factors to consider. Once you’ve decided on the type of trap, there are many...world, approximately 50% of predicted genes have unidentified roles as well (Vanni, et al). Is it true that...genes are truly dispensable or are the current identification methods missing something? Here we will review...review some time-tested gene identification systems that may have a leg up on their modern counterparts...just yet. Gene and promoter traps unambiguously identify the gene that they are monitoring as they are ...distance away from a gene which can make identification of the gene itself difficult. The best trap depends ...

...we can confirm the identity of the gene and check for any mutations. Once we identify the gene, we can ...talked about how our new Snapgene generated maps provide improved feature detection with an easy to use ...SnapGene for plasmid quality control are: To identify important plasmid features To align NGS sequencing...experiments for further plasmid verification Identifying plasmid features While our quality control process...in Snapgene’s extensive database. Snapgene also provides easy visualization of open reading frames, which...similar features and it allows you to more quickly identify the same feature in other plasmids. Aligning ...Aligning sequences with Snapgene    Once we’ve identified the features within a plasmid, we next verify that...

...need for target-specific antibodies and have been widely used across species and applications. In this blog... What are epitope tags? Epitope tags are short peptides introduced at the N or C terminus of a protein...purification or detection of the tagged protein. These peptides consist of as little as 8 or as many as several... (rabbit). Myc The Myc tag is a 10 amino acid peptide (EQKLISEEDL) derived from the protooncogene of ...immunofluorescence . However, protein purification is not ideal with Myc, as low pH conditions are required during...these species. HA The HA tag is a 9 amino acid peptide (YPYDVPDYA) derived from human influenza hemagglutinin... chicken) to this tag! GFP  GFP is more than a peptide – it’s a 238 amino acid protein that can also be...

...chance. Luckily, around the time I decided to look for work outside of academia, a job as a ‘Cancer Marketing...next and what options lie ahead of me? Like a considerable number of researchers, I had high hopes of securing...in the business of western blots and cloning, I decided to pack it in. I had just started month 8 of my...my colleagues and what I was researching, but I decided I needed a new challenge. Looking for sales and...yourself, sales and marketing positions like these are widely available, especially for life science researchers...purchased their products, so you have some of the best ideas on how to sell these products to other scientists...marketing knowledge. For most PhDs looking at careers outside of academia, sales and marketing can be great places...

...self-reflection necessary to identify areas that need work or growth. Here are some ideas for making your meetings...do you make the most of this new relationship? Consider adding formality and active goal setting to your...learned, especially noting action items that are identified. Keep a journal to record topics, plans, findings...you can find it in our Mentoring for Scientists Guide.  You can use it to clarify and discuss values. ...Attainable, Relevant, and Time-bound. It is a good idea to take larger goals (e.g. Figure out what I want...items and make progress. To illustrate this point, consider that I try to exercise every morning, but the ...postdoc can mentor a graduate student – you get the idea. There are many advantages to being a Mentor: Learn...

...promoter and the gRNA scaffold are provided with the kit. Oligonucleotides specifying the gRNA seed sequence...promoters. To use this plasmid, you simply order oligonucleotides for your chosen gRNA target sequences and ...uses Type IIS restriction enzymes, which cleave outside of their recognition sequence, creating flanking...Cas9 Golden Gate multiplexing is to clone the oligonucleotides specifying each gRNA target sequence into ...rectangles) are cloned into various plasmids using oligonucleotides. These plasmids contain Type IIS restriction...allows you to express 2-4 gRNAs, with four being the ideal number. First you generate four unique kanamycin-resistant...you to express two gRNAs constructed using oligonucleotide assembly.  Unlike some of the plasmids described...

...interaction allows scientists to easily purify and identify any proteins that are tagged with biotin using...biotin-phenoxyl radical in the presence of hydrogen peroxide. This radical reacts with electron-rich amino ...hours like BioID. APEX has allowed scientists to identify the proteomic map of the human mitochondrial matrix...multiple rounds of directed evolution until they identified a mutant with significantly increased sensitivity... binary protein-protein interaction as well as identify additional interacting proteins in one simple ...uses proximity protein labeling based on BioID to identify RNA-protein interactions (Ramanathan et al., 2018...monoglycine residue at the N terminus with a small peptide (LPETG). In EXCELL, the mgSrtA variant can be displayed...

...the gRNA (composed of a ~20 nucleotide targeting sequence and ~75 nucleotide scaffold sequence) is consistent... storm owing to its ease of use and utility in a wide variety of organisms.  While much of current CRISPR...being realized. There are a variety of reasons to consider using genome editing to change the genetic code...developed in other model organisms (see our CRISPR guide for common CRISPR principles). However, plant-specific...hereafter referred to as Cas9) and a single stranded guide RNA (gRNA) is sufficient to modify the genome of...nanostructures (Zhang et al., 2019) or cell-penetrating peptides (Santana et al., 2020) and plants viruses (barley...deliver CRISPR-Cas9 to plant cells and should be considered alternative to PEG or agrobacterium-mediated ...

... you can feel lost. Use this post to explore the wide range of careers available to scientists and open...from Grad Student to Entrepreneur Science careers divided by the most pertinent skills required Research ...and carry out in-depth research is applicable to a wide range of industries and is a highly desirable quality...including: Science Reporter Scientific Sales Epidemiologist Scientific Recruitment Meteorologist Research...explore the different options available and then decide on a course of action. Instead of rushing in, you...into a career, do your research Once you have identified possible careers, the next step is to research...computer programming skills? Other things to consider are wage, opportunity to advance, and how many...

... between these methods is that both attempt to identify new players in a biological process. Players can...approaches can be found. As an example, say you are considering either a cellular screen or a selection. A selection...molecules/drugs. With the advent of CRISPR, genome-wide knockout screens are possible with gRNA libraries...specific to your scientific question. Things to consider when selecting an assay system include: How specific...t yield any hits. Make sure you plan ahead and consider these questions to save yourself a headache later...population. For these experiments, the goal is to identify manipulations which either enhance or suppress...scene, there are other options which are worth considering. RNAi screens are similar to CRISPR, but instead...

...experimental needs, and can transduce (infect) a wide range of cell types with a high efficiency. However... part of the host genome that can replicate and divide along with the endogenous DNA. AAV also has low...AAV is also a cool tool in the laboratory! It is ideal for whole animal administration, such as in mouse...alternative virus, or other strategies may need to be considered if size constraints are an issue. Adenovirus...hamsters, although no cancer-connection has been identified in humans to date. Over fifty serotypes of human-infecting...human-infecting adenovirus have been identified so far! Adenovirus generally has high immunogenicity, ...viruses used in biological research, you might have an idea which ones could be helpful to you. Interested in...

.... Manufacturers rarely provide the construction plans or CAD (computer aided design) drawings of their...users the chance to understand the "black box" provided by the manufacturers and enables customized experiments...limitations into account in the development process. Widely used, commercially available tools such as the ... the community to regularly replicate designs worldwide. Distributing hardware via globally established...MIT), and documentation (e.g. CC-BY) allow users outside the project to modify the raw and design files,...of work from universities and research centers provides a potential conflict between traditional IP-based... for innovative organizations and companies to provide problem-solving platforms.     Fig. 3: ...

...team of twenty excited undergrads. Team IISER-B’s idea for the iGEM 2019 is to take bacteria out of their...objective is to induce tolerance towards cold, we decided to focus on a special class of enzymes expressed...usually defined between a fairly nippy 20°C to a decidedly uncomfortable 45°C). Their more adventurous cousins...fruit juice clarification, meat tendering, and oligosaccharide synthesis. A major incentive to use cold-active...industry as it can potentially help minimize the incidental losses like spoilage and improve the productive...inefficiently that no industrial operation would ever consider them worth the trouble.  Enter ELSA: E. coli Learning...Acclimatisation To resolve this impasse, our team decided to create a hybrid system where we could incorporate...