...other RNA components are required? CasPEDIA provides information on crRNA and tracrRNA requirements, along...field has exploded with dozens of Cas enzymes and variants available. Without a comprehensive resource, it... and cleaves ssDNA in trans. In sum, CasPEDIA documents the target substrate (DNA/RNA and strandedness...(nick, bind, cleave, mixed). This information represents the ‘big picture’ features of the enzymes documented...a candidate Cas for your experiment. Target requirements As we all know, a Cas guided by a gRNA is useless...) in some cases. The resource displays these requirements in regard to their sequence (NGG, etc.) as well...experiment. Perhaps you are a structure junky who just wants to learn about Cas domains. CasPEDIA either directly...

... interested in using GCE, here are the basic requirements of use. First, and most obviously, you need ...incorporated ncAA, ready for you do to any number of experiments with. The catch The rapid expansion of GCE led...That many GCE technologies have idiosyncrasies presents a significant barrier for those wishing to use...GCE aren’t able to successfully complete their experiments and many others are dubious enough that they ...optimization parameters the same for all three components, meaning that the final protocol may involve ...’s not uncommon to find protocols that require reagents which aren’t broadly or easily available, so you...protocol that you’ll have to rework to include reagents accessible to your lab. The solution GCE provides...

...the most recent advances and how some of these variants may be appropriate for your specific research ...nuclease When choosing the Cas9 nuclease for your experiments, there are three important factors to consider...isolated from different organisms and engineered Cas9 variants that were able to target different PAM sites (... be targeted (Hu et al., 2018).  These altered variants can allow for targeting of your genomic loci of...sensitivity in choosing the Cas9 to use in your experiments   The CRISPR drawer. So many options! ...off-site activity of Sniper-Cas9 to different Cas9 variants using gRNAs of different lengths (Lee et al., ...suitable for in vivo research and potentially treatments. Cas12b, characterized by the Zhang (Strecker...

...domain from PE6e-g produced additive improvements. These variants are worth testing if you want to make...Over 75,000 pathogenic genetic variants have been identified in humans and cataloged in the ClinVar database... potential to correct only a minority of those variants in most cell types. But prime editing, a CRISPR...goal of the experiment can all impose different constraints and affect how you select and design your prime...research, the Liu lab created and evaluated 19 PE1 variants with RT mutations known to increase activity, ...system as complex as prime editing, every efficiency counts! The optimized enzyme can be used with any of the...PE2max, PE3max, etc. PE6: Specialized prime editing variants Four years after their initial publication, the...

... of the party goers, the piñata bursts and its contents spill to the ground. There is something akin to... trouble is, with so much choice, in those few moments after the piñata burst, value was subjectively ...disconnect arising between those reviewing the contents of the piñata and the audience blindly receiving...proceed in the future. When we think of designing experiments using CRISPR, there are two major areas where... The foremost handles the design of sgRNA and represents the lion’s share of the currently available tools...example is CRISPR-GA. These tools assess repair events by type (NHEJ/HDR) and track indels at a single...more common. User interaction: Software design elements that fall into this category describe how the ...

...bind to the operator. The bound repressor then prevents transcription from occurring, which means that...), TetR preferentially binds Tet over the TetO elements, allowing transcription to proceed (pJKR-H-tetR... virus. tTA binds to promoters containing TetO elements (often linked in groups of seven as a Tet Response...binds tTA, resulting in a confirmation change that prevents binding to the promoter and turning transcription... Bujard, both the promoter and transactivator components of the system have been engineered and improved...Tet-Off vectors (Tet-Advanced)” are the most recent variants of the Tet-Off system.  For more information on...binds cTA, resulting in a confirmation change that prevents binding to the promoter and turning transcription...

... can start your experiment? What about all the reagents you need to order? Sometimes, it feels like an... Doench also calculated the cost of consumable reagents used to make a plasmid, which came out to $115...means that the same plasmid would cost $155.25 in reagents and consumables today. Finally, this doesn’t account... the scientists designing and performing the experiments. This cost varies hugely based on a number of...assume it’s at least equal to the $155 spent on reagents. Five ways to decrease plasmid production time...plasmids with high-throughput methods that save on reagents, consumables, and time. We also handle the MTA...order to preserve time and money for their own experiments. Keep good records Oftentimes, you’ll be able...

...organize events that bring different voices together to talk about genetics. In addition to events that we...improved understanding of how our bodies work, new treatments for debilitating diseases, and maybe even ways...individual consumers of technologies and as societal participants in the political process. My role at pgEd I ...readership, and working with middle and high school students. One of these organizations was pgEd. After obtaining...industry conferences, as well as schools, community events, churches or mosques. On occasion, we also get ...researching and writing – following the latest developments in genetics research, their applications and...landscape of genetics and our consistent stream of events mean that there is never a dull day in the office...

...measure these properties, and what fascinating experiments could push these tools even further? All of these...and find further tips for using FRET in your experiments here.     What is this FRET you speak of? Förster... acceptor In order for FRET to occur several constraints must be met. The donor and acceptor fluorophores...improved dynamic range for fluorescence lifetime measurements." PloS one 10.8 (2015): e0134436. PubMed PMID...the probe. More on these biosensors later on). Experiments using intermolecular FRET probes are typically...reference standards can be used to validate FRET measurements and serve as a type of positive control. In ...questions about cellular processes. Currently, FRET experiments can probe protein-protein interactions, measure...

... Figure 1: SARS-CoV-2 proteins and other components. Image from Maya Kostman for the IGI.     ...COVID-19 research in other labs. Spike protein, variants, and pseudotyping The SARS-CoV-2 virus depends...cellular receptor. To more easily study the spike variants, scientists have developed ways to pseudotype ...lab. Jesse Bloom’s lab published a protocol and reagents for pseudotyping lentivirus with the SARS-CoV-... has had many opportunities to evolve. Several variants are now circulating and plasmids containing these...pseudovirus and those encoding different spike protein variants that can be incorporated into pseudoviruses. The...at neutralizing the different SARS-CoV-2 spike variants. You can learn more about their research in this...

... but these reagents and strategies are not available to those working with yeast or plants. Using MoClo...the basics of what MoClo is and what types of experiments it enables. What is modular cloning? Modular ...assembly. Simply put, it assembles individual DNA components into larger units or circuits with functional...and a ligase are used to assemble multiple DNA fragments together in a one-tube reaction. Even though most... test a variety of different orientations and components within the system to maximize production potential...multiplexed gRNA systems as well as additional components such as selectable markers, promoters, enhancers...is especially useful for more complex CRISPR experiments (e.g., many guides/parts delivered) in certain...

...the risk of such events, newer lentiviral packaging systems divide the viral elements among multiple plasmids...Replication competence can arise from recombination events during viral production and poses a biosafety risk...carrying the transfer gene. By dividing the viral elements across multiple plasmids, the risk of recombination...for transfection can make these optimization experiments a bit more challenging. With these considerations... thereby increasing the number of recombination events that are required to generate RCL. To overcome ...drive high levels of expression of certain viral components; according the manufacturer these changes result...lentivirus, the plasmids encoding the structural elements, envelope, and transfer gene are transfected into...

...decisions. In my work as a thesis coach for graduate students, I heard about supervisors in all of the above...also considering the needs of other people. Some students and postdoctoral fellows are hesitant about expressing...help you to become more assertive and resolve disagreements with your supervisor, even if he or she is a...will meet both of your needs. 6) Put important agreements in writing One of the most frequent sources ...approximate timelines. Be sure that you keep your commitments, and if for some reason you are not able to, ...they can count on you to follow through on your commitments. Assertiveness is a skill that will help you ...supervisors might try to intimidate you with negative comments (hostile-aggressives, complainers, know-it all...

...with different fusion proteins for different experiments over the years — a green fluorescent protein,...choose different colors or ligands for different experiments without having to clone a whole new construct...Live-cell labeling strategies. The green structure represents a fluorophore or other useful ligand, gray structure...HaloTag is actually HaloTag7, and recent specialized variants run up to HaloTag11. HaloTag is now the most popular...the results right away. Or perform pulse-chase experiments by sequentially adding different ligands to the...between different colored ligands at different time points.   Figure 3: Single-molecule tracking of...assembly-regulating proteins in vitro reveals complex events. a) Micrographs of a fluorescently-labeled actin...

...technologies have transformed the palette of experiments possible in a wide range of organisms and cell.... elegans approaches rapidly enrich for editing events without the need for any selective marker to remain...the awesome power of genetic selection. Knock-in events tend to be rare, and the challenge with any experiment...in any genetic background, but require variable amounts of experimental manipulation and screening.  A ...screening 768 F1 animals). Recovering rare editing events in a sea of unedited animals struck me as a “needle...into the homologous recombination repair pathway. Reagents required to perform pha-1 co-conversion are available...knock-ins (Ghanta et al., 2020). Together, these improvements from the Mello lab resulted in strikingly high...

...education? Let us know about your educational events in the comments section below! Additional Resources Visit...Last week, 20 students and four chaperones from Denis-Yarmouth Regional High School visited our labs ...Learning about careers at Addgene Afterwards, the students split into two groups for an in-depth look at ...Biology Lianna Swanson was very popular, with the students excited to see a real working lab full of busy...Operations Coordinator: “Kory was awesome!”. The students also got to learn about intellectual property ... technology was a rousing success! Many of the students were interested to see so many different career...such a great day, we look forward to hosting more students next year! Does your lab or organization do work...

...portion of the plasmid that is important for your experiments. There are a number of factors to consider when...

...majority of the respondents (70%) used a lipofectamine-based method to deliver NgAgo ingredients into the cell... questions about the NgAgo protocols used by respondents (transfection method, amount of each component...successful indel formation at a target site. Also, 41 respondents attempted to use the technique for epitope tagging...Survey The take-home message While most of the respondents have been struggling to achieve genome editing...generation and epitope knockin was achieved by a few respondents suggesting that NgAgo can indeed be optimized... take-home message thus far, based on survey respondents "Final Verdict", is that more time is required...Addgene Blog - Leave suggestions and tips in the comments section below or contact [email protected] if you'd...

...Spiking your media with supplements such as magnesium salts, buffering agents, and/or supplying additional...Use a strain optimized for plasmids with repeat elements, such as viral plasmids with inverted terminal... and prep high quality DNA that contains these elements. Be aware of endA+ strains Strains like Stbl3 ...including HB101 and derivatives, release large amounts of carbohydrates that can inhibit the cell lysis...of protein, not plasmid DNA. If you need large amounts of DNA from a plasmid in a protein expression strain...culture media Changing to a richer media or adding supplements can improve your cell density, leading to an ...normally recommended antibiotic concentration. Add supplements to the media You can achieve higher cell density...

...phenotype can lead to a wealth of additional experiments investigating the role of individual genes or...carry out so-called “forward genetic screening experiments”, it is certainly the most robust. In this blog... genome-wide screens and highlight some of the reagents that have been made publicly available through... and versatility. CRISPR/Cas9 consists of two components: the non-specific endonuclease (Cas9) and a single...72005145@N00/5600978712/ What CRISPR-based reagents are available for genome-wide screens? The goal...gRNA-containing plasmids. CRISPR genome wide-screening experiments commonly use lentivirus to deliver a pooled population... mind that libraries are only as good as the experiments you use them for! A well-developed biological...