...the CRISPR toolbox. We'll cover how Cas13a was identified, the structure and function of Cas13a with a ...cleaving CRISPR nuclease Cas13a was originally identified in 2015 (Shmakov et al., 2015). They were using...acquisition following infection, as a form of “bait” to identify new CRISPR-associated proteins within the bacterial...bacterial genome. From this analysis, they identified 53 potential candidate genes that fell into 3 categories...Common CRISPR Enzymes Name  Enzymatic Domains Guide RNA Target PAM (PFS) Cleavage Mechanism Cas9 HNH...requires that each DNA target have high sequence identity to the spacer sequence and contain the appropriate...Cas13a as a diagnostic tool. A pool of DNA or RNA nucleotides containing a sequence of interest (red) is amplified...

...Viral vector topic page Beginners guide to viral vectors Quick guide to all things lentivirus Resources...Resources on Addgene.org Gamma-retrovirus Guide Lentivirus Guide ... or miRNA. The ever expanding CRISPR field makes wide use of retroviral delivery systems, especially lentiviral...functional screening of thousands of genes in genome-wide experiments. Gamma-retroviral vs. lentiviral vector...gamma-retroviral vectors in our Gamma-retrovirus guide and find popular packaging and gamma-retroviral ...Lentiviral Systems can be found in our Lentiviral guide. Popular packaging and lentiviral envelope plasmids...VSV-G, may be used across both systems. Below I provide a brief overview of a few popular gamma-retroviral...

...genes we’ve tagged are widely recognized markers of cellular structures, we provide guidance on how to make...in creating the plasmids was to identify the gene of interest and decide which terminus, N or C, to tag...structure of interest. Endogenous gene tagging is an ideal solution because it allows for tagging and visualization...plasmids that use CRISPR/Cas9 to endogenously tag a wide variety of genes with GFP. These plasmids are available...plasmid that contains long stretches of DNA on either side of the fluorescent protein sequence that is homologous... being inserted: one thousand base pairs on each side, in fact. Later, when we transfect the cells, these...GFP tag and 1kb of homologous DNA flanking both sides (about 2.7kb total)—is inserted into a plasmid backbone...

...this post to guide you through them and make the best use of all the information provided by your colleagues...depositors have provided primer sequences for use, these are listed.  We also provide a link to our list...this plasmid from our search page (check out this video for a walkthrough of the ways you can search our...flames about? Flame logo: The flame that appears beside some of Addgene’s plasmids is an indication of ...their own labs. We highly encourage depositors to provide plasmid maps whenever possible. GenBank files can...research! Sequence information: Depositors may provide full or partial sequence information for their ...reactions (‘Addgene Sequences’).  These sequences are provided in FASTA format (example) and can be analyzed ...

...2020 by Marcy Patrick. CRISPR technology has been widely adopted for genome editing purposes because it'... your next experiment! In this blog post we’ll provide an overview of some CRISPR mammalian expression...and potential delivery methods.   General considerations for planning your CRISPR experiment As with...experiment, there are many factors that need to be considered during the planning process. For CRISPR experiments...choose between them? One of the first steps is to identify what CRISPR components you will need to deliver...deliver a donor plasmid or single-stranded DNA oligonucleotide, so you will need to make sure your expression...are compatible with all your components. Next, consider the best form for those CRISPR components based...

...wasn’t exactly my idea of a dream job, but I knew that I enjoy helping people and decided to compromise and...also explore a range of alternative careers outside of acidemia to have the best chance of finding their ...variety of different organizations, so I tend to provide them with an overview of my experience and how ...about the different positions to enable them to decide for themselves.  Here is my experience looking ...language in Spanish or French, and so they were ideal candidates for these positions.  I had to reassess... to university to study a Masters of Science I decided to apply to study a Master of Science (MSc) in ...research project was hard; I was researching the epidemiology of breast cancer and I loved the practical aspects...

...can also be exciting! Identifying a host institute or lab So, you’ve identified a gap or need in your ...collaborations Transferable Skills Guide: External collaborations Transferable Skills Guide: Teamwork      ...or maybe both, a visiting scientist stint may be ideal for you. Here, we will chat about who might want...visiting role is because the host lab or institute provides something that you can’t obtain at your current...in. Visiting a lab that has this expertise will provide you with guidance and training so that you yourself...new institute and mentor that they may want to consider for future work placements. The chance to learn...your research and decided you want to pursue a visiting position. What now? First, you’ll need to find someone...

...'s a good idea to bounce your ideas off of senior faculty members who can help you identify overly ambitious...the central idea. It should be at a high level, broadly understandable to scientists outside your field...least a few times with people that are outside your lab and outside your field. If they can understand and...?" is an open-ended list of proteins. Instead, consider "how do proteins X and Y regulate the Z pathway... or your data favor a different model. Finding evidence for or against a model is a standard scientific...coherent unified story and model can help make your ideas easier to understand and more exciting. Practice...that is easily visible from the back of the room. Consider going beyond the whiteboard. You may wish to create...

...some 1010 oligonucleotides could be screened for their affinity to a wide range of non-nucleotide targets...over the course of the next two decades, these nucleotide-based ligand binders would prove to be highly...their broad range of targets, including proteins, peptides, amino acids, drugs, metal ions, and cells (even...such as a protein. Such flexibility makes aptamers ideal candidates for biosensors. Aptamer biosensors are...several different ways. Researchers have used oligonucleotides in fluorescent visualization techniques for...relatively bulky. One of the advantages of oligonucleotides over proteins is their modularity, which is...- bypassing translation altogether with an oligonucleotide-only sensor could save precious minutes in ...

...why we decided to do an AMA in the first place and give you some reasons why you should consider using ...hope I was able to provide some good resources to the basic questions and to provide a small bit of support...interested in selling a product than communicating your ideas with people or discussing science, it is a great...users can participate in forum discussions about a wide variety of topics. Individual forums are hosted ...STEM, work-life negotiation, and mentorship, we decided to have her participate in an AMA on these topics...substantial traffic that we thought it would be a good idea. After a bit of research, we realized that the r...You can check out the r/Science AMA Submission Guidelines here, but essentially we just needed to send ...

...science policy even is! In this article, I hope to provide a brief explainer of the who, what, where, when...The folks who work in science policy come from a wide variety of backgrounds with technical expertise ...creatively connect seemingly disparate individuals and ideas toward solving one problem. It often requires thinking...the field.  Read the CCST Science Policy Career Guide to learn more about science policy as a profession...in DC, is that so much policy actually happens outside of the government itself. You can work to influence...communities. Think a university system who develops evidence-based university policy to combat sexual harassment...harassment in academia or a large company who decides how they are going pursue regulatory approval for a...

....  These primers are typically ~70 nucleotides long (20 nucleotides that anneal to the DNA sequence of... Frequency Special Considerations dsDNA Insertion or deletion of >20 nucleotides 1 out of 104 to 105...tube. The donor DNA substrate only requires ~50 nucleotides of homology to the target site for recombination...insertions or deletions greater than approximately 20 nucleotides, while ssDNA substrate is best for point mutations...of interest and 50 nucleotides of homology to regions flanking the target site).  The best applications...out of 104 to 105 colonies. Pro Tip: Use a high-fidelity Taq polymerase with proof-reading ability for ...substrates can be generated by ordering synthetic oligonucleotides or denaturing short PCR products. Either way...

...easily and precisely identifies plasmid features that differ by only a few nucleotides, such as the fluorescent... BLAST to verify plasmids. Analyzer guide A quick guide to all of the tabs listed above and their...Feature Library - SnapGene’s software seamlessly identifies a variety of common plasmid features such as ... Enzymes, and Primers - The new maps have the identities of most features labeled directly on them making...making them much easier to quickly identify. Improved Restriction Enzyme Handling - SnapGene’s software..., & downloadable .gb & .dna files Once you’ve identified a plasmid that you want to learn more about, ...your sequence viewer of choice. If the depositor provided a custom annotated GenBank file, this can be found...

...Fab Fragment? Consider the classic IgG antibody (Figure 1): it is composed of four peptide chains (two ...two identical heavy chains and two identical light chains) each with a constant region and a variable region...chains are covalently linked to one another by disulfide bonds between the two heavy chains and between...four chains make a Y-shaped protein that can be divided into the Fragment Crystallizable region (or the... means that it is relatively easy to physically divide up the different regions, both through genetic ...proteolytic digestion that retains an antibody’s disulfide bonds within the hinge region. The resulting product...')2 fragments, which reduces the hinge region disulfide bonds, resulting in two independent Fab fragments...

...amino acids or peptide tags. 5) Strong protein-protein interactions with other peptides. Created with ...Addgene’s antibodies are provided in a buffer with the antimicrobial agent sodium azide, which can interfere...spectral properties of the fluorophores you are considering to make sure that they’ll be compatible with ...antibody conjugates (especially if you start to consider clinical applications). But there are a few other...we would be remiss not to at least mention. Oligonucleotides allow for sensitive detection of low abundance...multiplexing potential. You will find antibody-oligonucleotide conjugates used in assays like proximity ligation...strong protein-protein interactions with other peptides, like an Fc-binding domain (Figure 1).  ...

... Other useful backbone modifications to consider include amide linkages, unlocked nucleic acids, and a...to simply swap out several of the ribonucleotides for deoxynucleotides. A partially DNA gRNA is surprisingly...perfect guide; now check out our blog posts on choosing the best Cas9 enzyme to pair with your guide and ...larger CRISPR RNA (crRNA). In addition to the 20 bp guide sequence, the crRNA also contains a ~20 bp repeat... a constrained ethyl.  Can’t decide which RNA backbone modification is best? Studies suggest coupling ...use in applications like siRNA and antisense oligonucleotides. Some of these synthetic modifications, specifically...photoactivatable and photocleavable gRNAs. Photocleavable guides are generated by introduction of a single photocleavable...

...previously induced gene knockout. Besides amine- and DNA-binding dyes, nucleoside analogs like bromodeoxyuridine... useful for classifying and sorting cells by identifying lineage markers, like many of the cluster of ...one or several of those techniques can be used alongside standard surface labels. Here, I will introduce...streptavidin conjugates are commercially available for a wide range of fluorophores, allowing for great flexibility... you to include further (non-lineage) markers alongside, e.g., for cellular activation. In addition, FACS...interest is not located on the cell membrane but inside the cell. Due to their chemical nature, antibodies...antibodies to enter the cell (Figure 3). A useful side effect of the fixing/permeabilization (fix/perm)...

...mutagenesis of Spinach. In doing so, they identified the nucleotides that contribute to the instability of...describes the enrichment process used to evaluate oligonucleotides for binding affinity to a specific target,...molecule derivative of GFP called 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI). DFHBI is membrane ...studies. Additionally, Spinach, the first aptamer identified to bind DFHBI, is highly unstable in vivo due...Spinach. It was developed in a study of artificial guide RNAs that can be used to simultaneously target cellular...Broccoli, the other green fluorescent aptamer, was identified through directed evolution. First, a pool of ... that activate the fluorescence of DFHBI were identified using systematic evolution of ligands by exponential...

...in the face of evidence” (4). Frustration among scientists has been rising as evidence of climate change...not in the job of having evidence”, scientists want to make sure having evidence is the job of everyone ...marching for every cause. The references provided also only provide a snapshot into the opinions or research...Health endanger scientists’ ability to uphold their side of the bargain and do the research required to inform...while alienating the other While scientists do identify as Democrats more often than Republicans, the ...Republicans (16). This is a Pro-Science and Pro-Evidence march. Who is marching and why? Who? All sorts...include science in decision making, it will be considered a success. Below are resources for the march ...

...attribution: Zetsche, Bernd, et al. "Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system." Cell... cleaves in a staggered fashion, creating a 5 nucleotide 5’ overhang 18-23 bases away from the PAM. Cpf1...through homology-directed repair. Both Cpf1 and its guide RNAs are smaller than their SpCas9 counterparts,...smaller than any of these options (2.7 kb with 24 nt guides), so it has the potential to eclipse any of the... al. and Kleinstiver et al. characterized genome-wide editing efficiency of two Cpf1 orthologs known to...orthologs was only slightly lower than that of the widely used SpCas9 and comparable to SaCas9. As seen with... with Cas9 orthologs, Cpf1 efficiency varies widely with gRNA sequence. Both groups used multiple methods...

... dedicated summary page with PubMed identifiers and links provided, in addition to a list of individual...read-outs other than cell viability. The following video provides an in-depth walk through of how to use ORCS...and other species. By accurately compiling genome-wide datasets, which are often obscured in large supplementary...different datasets that might otherwise be hard to identify. Figure 2: Users can cross-interrogate ...linked to the original publication via its PubMed identifier, which can also be used to search the entire ...as the original quantitative gene-level scores provided by authors rather than sgRNA-level sequence read...tables available with the publication are also provided for the user. This approach serves to maintain...

...and are inconsistent between universities!) This guide tells you how to use online platforms, such as Zoom...recognizing the importance and benefit of remote and video communication. But first, take a deep breath; at...navigate a global pandemic can also be the thing that provides a new perspective that helps to clarify the harder...before selecting an advisor, it is always a good idea to introduce yourself to a few members of your desired...incredibly useful, as it gives a more comprehensive idea of that advisor’s research interests which can be...further discuss aspects of the lab, research, and provide them with a CV or transcripts if requested Thank...Thank them, and eagerly await a reply. It’s a good idea to email around three professors per program. Most...

...they can’t be avoided. What are off-target effects? The CRISPR-Cas system is an RNA-guided nuclease that... gene expression by various mechanisms. The RNA guide is typically 20 nts and the genomic locus must often... concern, or nothing to worry about all. Let’s consider several scenarios below for a CRISPR experiment...An off-target rate of 5% is far too high when considering human patients and the diseases associated with...off-target measurements at those sites to get an idea of how high the off-target frequency is (more on...many more. While such software typically only considers DNA sequence and doesn’t take into account chromatin...the genome to reduce off-targets, it is time to consider your Cas enzyme. Even if there are similar sequences...

...donor oligonucleotide can be electroporated into the mouse zygote (Qin et al., 2015).  When inside the ...However, if repair material is provided (in the form of an oligonucleotide or plasmid), precise changes ...genome, the mouse genome is made up of 3 x 109 nucleotides (nt), and encodes 23,000 or so genes. It would...for genome editing in eukaryotes, with the most widely used CRISPR genome editing system derived from ... CRISPR/Cas system makes use of 3 components, a guide RNA (gRNA) of about 125 nt that specifies the target...break (DSB) at the target site, and a donor oligonucleotide or plasmid as the repair material if needed...create a mouse model, the gRNA, Cas9, and donor oligonucleotide or plasmid components are brought together ...

... around the targeted nucleotide. Introduction of guanine-mismatches in the guide across from these off-target...CRISPR field. Just a year after Feng Zhang’s lab identified Cas13a (C2c2) (Abudayyeh et al., 2016) as a RNA-targeting...E488Q). They also adjusted the structure of the guide RNA by introducing a C opposite the target A to ...editing with various spacer lengths from 30-84 nucleotides, and designated this system REPAIRv1. Using next-generation... see if they could shrink the construct. They identified ADAR2DD(delta984-1090), which decreases the REPAIRv1... off-target editing. In one direct transcriptome-wide comparison, REPAIRv2 reduced off-target sites from...They note that RESCUE is most active in a 30 nt guide when C or U base-flips are present across from the...

... targeted locus on either side of the new sequence. This homology will guide the cell to use the donor...the first questions are where, which, and what. Identify where in the genome you would like to introduce...exogenous DNA can be used as well. Below are some considerations for designing donor DNA for successful HDR ...on the size of the knock-in. Single strand oligonucleotide donors (ssODNs) with homology arms as short...need to be used due to synthesis limits of oligonucleotides. These donors traditionally have larger homology... site until either the PAM site or part of the guide’s recognition sequence is destroyed via mutation....introducing a mutation in either the PAM or the guide recognition sequence in the donor DNA, you can guarantee...

...relative proportion of guide RNAs (gRNAs) across the population. Recall that guide RNAs are the targeting...’ll need something like Google Maps to help us identify and visualize addresses. That’s where enhancers...three-step approach for designing an experiment to identify the critical NANOG binding sites: 1. Find Enhancers... 1: (H3K4me1 + H3K27ac) – H3K4me3 The following video demonstrates how anyone can find enhancers with ...genetically programmed to CRISPR out the enhancers we identified in step 2. The result is a heterogenous population...to create targeted disease therapies with minimal side-effects on healthy cells and maximal effect on unhealthy... combination with the techniques from Table 1, provides us with powerful GPS-like techniques to explore...

...innovative ideas in all facets of the entrepreneurial process in order to develop those ideas into commercial...excellent people with mediocre ideas than mediocre people with excellent ideas. I am lucky to have launched...self-knowledge and an unwavering devotion to seeing your ideas become something tangible.  When you think of entrepreneurship...company, you need to determine whether or not your idea is viable.  Go to experts you trust and pitch what... received from our network.  We started with the idea of a biotechnology investment fund that would apply...our early stage.  After each pitch we made, we considered the feedback and adjusted our business model ...You’ll find that no matter how well you know your idea, you will rarely succeed in launching a company ...

...Trends: Most tools provide tabular HTML formatted output. Surprisingly only 2 tools provide FASTA output. ...success to technical expertise, thoughtful single guide RNA (sgRNA) design, and the abnormally low GC content...OMICtools. It must be confusing for a newcomer to decide on the right software to use. With so much choice...sgRNA design tools The goal of this post is to provide some insight into available CRISPR software tools...qualities of what determines good sgRNA design. Incidentally, the first software for sgRNA design (by Hsu...scoring algorithms and parameters. This tapestry of ideas is what makes choosing the right tool confusing ...functions are not entirely necessary but should be considered extremely useful, depending on design goals. ...

...resistance to pyrazinamide (PZA) (a drug commonly used to treat tuberculosis) and in identifying new drug ...wild lettuce. Aside from their use as food, infusions of L. taraxacifolia leaves are widely used to treat...Ersing Seeding Labs and Addgene collaborate to provide scientific resources, equipment, and plasmids to...specifically, the Addgene-Seeding Labs plasmid grant provide plasmids to these researchers. In the third round...control. In her current research, Sheen aims to identify new genes related to PZA-resistance by using different...using CRISPR on our blog). Her research should provide us with a better understanding of PZA action and...treat several diseases. Her goal is to identify and isolate pure plant compounds with antidiabetic properties...

...few minutes. The host could also set aside Breakout Rooms to divide the audience into small sessions and...To date, aside from doing science, he has assisted many researchers in gaining the confidence to launch... at the lab meeting, the team can discuss those ideas and build from those.   Figure 1: Virtual...focus on. Use collaborative cloud apps It is a good idea to use collaborative cloud tools such as Google ... and Sheets during Zoom lab meetings to chip in ideas and take minutes that can be accessed by anyone ..., where everyone stands up, disappears from the video, and prepares a coffee, or simply moves away from...I believe this is a great functionality that is widely used and could bring back a taste of that casual...

...Cas9, for which efficiency varies widely across guide RNAs, each guide tested with CasRx yielded >80% knockdown...Cas13d proteins they identified have little sequence similarity to previously identified Cas13a-c orthologs... to tap the wealth of CRISPR possibilities. To identify Cas13d, both groups used very general bioinformatic...independently process their own CRISPR arrays into guide RNAs. crRNA cleavage is retained in dCas13d and ...its low immunogenicity. But these studies also identified other advantages, including Cas13d-specific regulatory...produced very similar cleavage products even when guides were tiled across a target RNA, indicating that...Previous CRISPR exon-skipping approaches used two guide RNAs to remove a given exon from the genome, and...

...ongoing Plasmids 101 series. Plasmids 101 will provide you with an overview of general molecular biology...plasmid with an insert? Blue-white selection is a widely used method to do just that! (To read about other...lactose analogue, IPTG (isopropyl β-D-1-thiogalactopyranoside). (To be completely accurate, IPTG binds...x-gal (5-bromo-4-chloro-3-indolyl-β-D-galacto-pyranoside) into galactose and an insoluble blue pigment...encoding this section of amino acids (called the α-peptide) to a lacZΔM15-mutant bacterial cell in trans complements...engineered a multiple cloning site (MCS) into the α-peptide (represented as an orange wedge in the figure on... your DNA (red) is cloned into this MCS, the α-peptide gets interrupted as shown in cell A, and thus will...

...relationships outside your organization. The mentor in your own workplace is able to: Help you identify specific... and become an insider faster A mentor that works elsewhere is able to: Help identify the skills you... you meet, the more potential mentors you will identify. Finding potential mentors is one of the many ...done this with people I work with and people from outside my work for almost 20 years. It is good to be a... this recent blog 5 Strategies for Finding Your Ideal Mentor. One of the best was to notify your network...family and friends know you well and might have good ideas for who you should connect with. Listen to our podcast...you will need to make a change Provide perspective on core values at another company/department Give more...

...nonprofit institution, you can order plasmids covering a wide range of fields from Addgene. What you might not...this kit have been used to purify a variety of peptides including antibiotic efflux pumps and ankyrin ...followed by leader sequences (encoding small leader peptides) downstream of which you can clone your gene of...interest. Translation of the standardized leader peptide prevents secondary structures in the mRNA encoding...from resulting in the production of a downstream peptide, the transcription terminators in this series are...plasmids from this kit include the production of a suicide switch in cyanobacteria, synthetic inverters in...biotechnologically important microalgae: intrinsic suicide switch implementation in cyanobacterium Synechocystis...

...demographics and the information is often confidential (see image below). Slide of information gathered by Postdoctoral... this very idea that postdocs are acquiring training to become professors that is provided as an explanation...transferrable to other environments. Slide from Paula Stephan’s Talk (All slides can be found here). Importantly...information does not, however, prevent us from identifying problems with the structure of the academic research... research workforce, and we are beginning to identify potential solutions. Keynote speaker Paula Stephan...masters programs to prepare scientists for positions outside of academia Require that postdoc salaries be paid...coming months and welcome guest bloggers who can provide additional advice to trainees navigating the waters...

... BioID platform for identifying protein-protein interactions, but RaPID identifies RNA-protein interactions...ligase BirA* from BioID and allows a researcher to identify proteins that bind an RNA motif of interest in... like cross-linking immunoprecipitation (CLIP) identify RNA-protein interactions by cross-linking RNAs...using proteins as bait has limited utility for identifying novel RNA-binding proteins that interact with...RNA of interest. Current labeling methods are not ideal for in vivo work. BirA* requires 16-18 hours to ...Repository for Affinity Purification (CRAPome), to identify highly biotinylated background proteins reported... not well characterized RNA motifs. This bait identified a new interaction: binding of the RC2H1 protein...

... be identified using antibodies and immunohistochemistry techniques. Thus, these proteins provide a versatile...to the plasma membrane.   Our studies provided evidence that some fluorescent proteins can be, in ...facilitate its detection. Fluorescent proteins are most widely used tags because they emit endogenous fluorescence...versatile method to identify the expression of transgenes of interest. Furthermore, fluorophore variants...red fluorescent proteins) allow simultaneous identification of more than one protein of interest. However...were still cytoplasmic. Use of ‘small’ tags to identify proteins Given the limited expression of hM4Di‐mCherry...proteins when the fluorescence is not essential to identify the expression of the opsin or chemogenetic receptor...

... Addgene's resident blog editor, I could take the needed steps to continue where the guide left off - ...molecules can bind to a wide variety of targets, including proteins, peptides, amino acids, drugs, metal...I love Addgene's new Antibody Guide and not just because I helped create it. But while antibodies, as...its thorns and every affinity reagent has its downsides. These reduced-in-size options often have reduced...situations where a suitable antibody has been identified but a smaller or less immunogenic version is ...light chain regions, connected by a short linker peptide. The lack of constant region makes them less immunogenic...proteins formed by two Fab fragments bound by short peptide linkers. They are bispecific, with a lot of flexibility...

...experimental design considerations used in all behavioral studies. General considerations for behavioral ...should be avoided as this will also increase stress levels and affect behavior. A fact to consider: Rodents...behavioral testing As a general consideration for behavioral testing in a laboratory, a high level of experimental...fluorescent light bulbs, for example) that are outside the humans’ audible spectrum. The experimental ...gene expression in a specific brain area, avoiding side effects from such modification in other areas. An... particles are smaller and may be preferable if wider diffusion is required. On the other hand, lentiviral...targeted to a specific area. Some other technical considerations for stereotaxic injections are: Vector titer...

...go-to providers of materials needed for optogenetics. There are several important considerations with ...optogentics to answer a research question. The flip side of the optogenetics coin is materials science-based...dental cement (a similar procedure as implanting a guide cannula). A fiber optic patch cable is then connected...materials required for each [1, 2]. This protocol [3] provides, in exquisite detail, the various steps required...able to penetrate brain tissue a short distance so ideal implant placement should be about 0.4-0.5mm above...stimulation. The scatter effect is also an important consideration when preparing the implant itself, which brings...least 85% of the initial laser output. The same considerations apply to the polishing and preparation of the...

...with the guide RNA.   Non-target strand The DNA strand that does not base pair with the guide RNA. Despite...hypoxanthine (shown) is the nucleobase component of the nucleoside inosine. Created with BioRender.com. Although...ABEs are base transition editors. They adjust the side groups of DNA bases and leave the core ring structure...some cases, the same repair pathways that were considered problematic in the development of CBEs and ABEs...structure. In order to bind DNA, Cas9 requires (1) a guide RNA, (2) a complementary DNA sequence, and (3) a...strands called an R-loop (Figure 2). This allows the guide RNA to base pair with the DNA target strand, leaving...non-target strand unpaired and accessible on the outside of the enzyme. Figure 2: CRISPR R-loop. ...

...industry, nonprofits, and publishing. The livestream video from the event can be found below along with a description...reproducibility “crisis” on our hands and instead provide a brief conceptual framework for the causes of ...protocols and use one another’s reagents with the confidence that they aren’t simply doing an experiment wrong...Luckily, many organizations have been working to provide tools that make it easier to share in-depth information...working with a plasmid from Addgene, you can be confident that you’re using the actual tool that you ordered...research needs. Other companies like NEB and ATCC can provide you with additional validated reagents, and, with...Protocols.io, JoVe, and Bio-protocol all work to provide more descriptive and interactive protocols that...

...knockdown, or other factors, consider using one of the alternative, albeit less ideal, verification approaches...the results across runs. If the results vary considerably, then the high degree of variability in the ...replicates are distinct samples that are treated identically, used to assess naturally occurring random biological...assays have their own unique controls that must be considered (Burry 2011). Perhaps the most critical are those...wild-type and knockout samples are treated with the identical protocols that the experimental samples undergo...with its antigen or a small epitope-containing peptide to block the antigen-binding site. The sample is...detectable, it is likely due to binding interactions outside of the antigen-binding site.      Secondary antibody...

...designed small RNA, or guide RNA (gRNA). The gRNA conventionally contains a 20-nucleotide sequence that is ...Activators: A Practical Guide Additional Resources on Addgene.org Check Out Our CRISPR Guide Pages Find Validated...nuclease-null—in order to effect different perturbations. Consider, for instance, the challenge of attempting to ...interacts with a distinct gRNA that allows users to decide which Cas9 protein is directed to which target ... truncated such that it exhibits 15 or fewer nucleotides of complementarity to a target site, Cas9’s DNA...truncated gRNAs Importantly, the use of truncated guides has largely been demonstrated to result in decreased.... An additional benefit of the use of truncated guides is that they can be delivered into systems that...

... about my interests and my ideas all the time. Once, I had this crazy idea that I needed help with: wouldn... you make the most of the opportunities you’re provided throughout your PhD program.   Find people who.... They didn’t always know the best direction to guide me because they had no experience with my career... open mind and open ears. Everyone I talked to provided new data. Every new experience taught me something...troubleshooting.” Trying out new experiments and new ideas is nothing new to a scientist. Framing my efforts...efforts in these comfortable terms helped guide me through the process. As I mentioned, my graduate office...disappointed in the lack of participation, but I decided to focus on what the experience taught me. The ...

...as well as highlight some considerations for their use. Then, we’ll provide information about commonly...decision tree can help guide your choice on which systemic capsid to use. Factors to consider include the delivery...goes beyond such restrictions, you may want to consider a systemic capsid for your AAV.   Systemic capsids... definition of systemic capsids. After some consideration, we offer this definition: A systemic capsid... after intravenous administration, distributing widely throughout the organism to target large or diffuse...commonly used and emerging capsids to help you decide which ones may be suitable for your experiment. Like all...and/or its intermediate engineered precursor. Considering systemic capsids for your research? While systemic...

...disciplines. Cast your net wider, explore tangential subjects, and let curiosity guide your intellectual journey...thus feel grateful) and identify areas in need of improvement. These unrelated ideas might hold the key to...simple steps to welcome these unforeseen moments. Widening your search The first step towards inviting serendipity...ultimately enhancing your project's potential. Widening your search creates fertile ground for serendipitous...encounters that could spark unforeseen connections and ideas. Maintaining openness and looseness of spirit The... exploration of tangents or seemingly unrelated ideas. It's beneficial to carry a notebook at all times...confines of a predefined path, opening doors to novel ideas and approaches. Moreover, the ability to embrace...

...develop their career skills for positions both inside and outside academia. Inspiring future scientists At ... ever heard of a Freshers Fair? If you’re from outside of Europe, the answer is probably no, but these...be used as welcome events for new students that provide ways for students to network, find groups with ...come. Some Freshers Fairs can include fairground rides and other fun activities and even invite members...thought we’d take some time to introduce you to the idea of the Freshers Fair and some of the great biological...students so that they can help support each other and provide advice. The society organizes numerous recreational... high schools to promote chemistry degrees and provide advice to students who want to study chemistry....

...mentoring for instructors, and provides opportunities for students to exchange ideas and results with peers and...al., 2020). Many have interpreted this trend as evidence that the best teachers and mentors pass on more... Biochemistry at the University of San Diego), decided to take a different approach. His own most impactful...experiences happened in the lab, and he wanted to provide the same kinds of opportunities for his students...but one that left him overwhelmed. Therefore, he decided to adopt a more focused format, where all students...analysis, structural prediction, and bench work to identify regions of MDH that interact with citrate synthase...Meeting. Some of my students published their work identifying new genes required for stem and progenitor cell...