...! A “complex” system Cas3 is part of the Class 1 CRISPR family, which means it requires multiple Cas effectors...systems work using a multi-Cas complex called Cascade (CRISPR-associated complex for antiviral defense). Cascade...compositions of the Cascade complex. Created with BioRender.com.   The Cascade complex Cascade is an important... Cascade complex from CRISPR type I-E. Created with BioRender.com. Thanks to this complex, developing... settings — mostly a consequence of Cascade's complexity. Figure 1: Schematic of Class 1 CRISPR...component of the CRISPR Cas3 system. Most Cascade complexes are comprised of a combination of Cas5, Cas6, .... It was originally suspected that Cas3 moved a couple of kilobases down the strand of DNA, Cascade caught...

...constructs to allow people to, for example, knock out a gene with simultaneous complementation, with a knockout...enable other people to do that too. We don’t have the capacity to send tools out to people ourselves, but... but we’d really like for people to use them. The MoClo Plant Kit makes it really easy to clone genes ...but have comparable expression levels to things people do use a lot. The problem in plant science is there...similar stability to transcripts as the ones that people use all the time. Now there are many, so if you... those working in plants, including many things people already use. It makes the switch to Golden Gate... Golden Gate is a conceptual barrier. Sometimes people don’t know how to start. They might want to use...

... The simple answer is that sometimes it is necessary or desirable to control or look at multiple genes...together for complex genetic analysis in Drosophila or for precise control over multiple genes in synthetic...binary systems. In recent years, a number of more complex systems have also been developed which don’t fit...that transcription is now OFF.   We’ll look at examples of both natural and engineered repressible promoters...transcriptional activation domain VP16 taken from the herpes simplex virus. tTA binds to promoters containing TetO elements...in mammalian cells, this system has also been implemented in a variety of other organisms, such as sphingomonads...UAS-driven gene expression. To further increase the complexity of the system, researchers have used split GAL4...

...is a fast, multiplexable method that combines CRISPR and recombineering-based MAGE (Multiplex Automated.... Adding complexity, they found that they could also include a CRISPRi system for multiplexed modulation...functions in S. pneumoniae and can be used to generate multiple mutations simultaneously (Jiang et al. 2013). ...mutations and 66% efficiency for small insertions. Multiplexing of two targets simultaneously is possible with...pCRISPomyces-2 contains Cas9 and a gRNA cassette. The simpler system of pCRISPomyces-2 displays a higher editing...multi-spacer CRISPR array, CRISPR-transposons can be multiplexed. Transcriptional repression (CRISPRi) in bacteria...transcription by physically blocking the elongation complex from binding the DNA or extending the transcript...

...process such as primer and plasmid design. Examples:  “multiplex design”, “multi-method design”, and “single-method...deals in post-experiment quality control, a good example is CRISPR-GA. These tools assess repair events ...published 1 Jan 2013, no longer available), is a simple catalogue of reported sgRNA designs. The second...similar to EENdb than CrisprGE, but is also a good example of how database tools can be used to aid design...this category are the most common between tools. Examples: “single-target design”, “multi-target design”...considered extremely useful, depending on design goals. Examples:  “feature aware”, “SNP aware”, “secondary structure... common utility functions are batch design or multiplex features. However, tools aiding in primer design...

...translational, biomedical, or applied sciences), people conduct research to answer questions about how ...applied. The current pandemic serves as a striking example of the applicability and importance of research...you’ll see in a lab. Position Common tasks Principle investigator (PI) Runs the lab Writes a lot ...questions and your excitement will in turn inspire the people around you. Be proactive Rather than applying only...the best research experiences typically come when people actively seek out science that genuinely excites...experience more tailored to personal interests. Talk to people, ask questions, and dig deep Talk to your professors...into is really important.  As you’re talking to people, though, try to be conscious of who has your best...

... as the building block of our understanding of complex biological processes. Many tools exist to perturb...in the context of the biological question. For example, while gene knockouts have long been considered...silencing,” of any gene that contains sequence complementary to the dsRNA (Wilson and Doudna, 2013). RNAi... translation, or chromatin modifications of complementary gene sequences (Figure 1). As a result, gene... of interfering RNAs.   RNAi vs. CRISPR: Complementary techniques It is fair to say that CRISPR has...of the technique—all that is required dsRNA complementary to your gene or genes of interest to trigger...constructs that enable insertion of ~400 bp complementary to your gene target as inverted repeats (Figure...

...single-stranded and self-complementary adeno-associated viral vector genome titers by droplet digital PCR. Human... lot. At Addgene, AAV particles are titered by droplet digital PCR (ddPCR) using primers and probes targeting...specific QC experiments were performed on your lot, please contact us. Full sequencing of the Viral Genome...of the viral capsid proteins VP1, VP2, and VP3. Samples of viral preparations are subjected to polyacrylamide...proteins as a fraction of total protein present in the sample is also determined and used to determine purity...shown relative to the total protein present in the sample.   Sterility Viral vector preparations are ...Lentiviral vectors are titered using a lentiviral droplet digital PCR (ddPCR) titration protocol (modified...

...always tell people the best part of my job is getting to talk to so many different people! I communicate...skills: working with data, working with people, working with complex processes Arm yourself with knowledge...management, and how does it relate to science? In the simplest of terms, a software product manager helps ensure...group is building the software tools for the right people at the right time. To explain a bit further, a ...manager works with software developers to help implement solutions for the stakeholders. Finally, software...all fits in with science. Well, I’ll give you an example. At my previous company, Ginkgo Bioworks, our software...backgrounds, most did not. It was my job to translate complex lab operations workflows into a format the developers...

...splitting up a sample into thousands of physically isolated partitions (such as droplets or wells on a ...clones for complete functional knockout of your target gene. Mismatch nuclease assays are completely “blind...of target DNA molecules present in the original sample with a great deal of precision. This means digital...theoretically detect a single target molecule in a sample. Digital PCR assays for mutation screening Now...use aren’t that much different from conventional duplexed primer probe assays often used in real time PCR.... The drop-off probe is designed to perfectly complement the wild type sequence and is not capable of ...amount of both mutated and wild type target in each sample can be quickly calculated (details in [2]) and ...

... Figure 1: Example of yes-no flow plot of the (negative) control (left) or with the sample (right). Negative...designed your first flow panel, you’ll load your samples into the cytometer and see one of two results for...protein, a yes-no plot is the best-case result. For example, you have introduced a GFP protein into your cells...somewhere between a 104–105 signal intensity. If your samples and controls were processed the same way, the GFP...Negative and positive samples are clearly distinguishable as two separate populations. Created with biorender.com...biorender.com. Pro tip! If your samples and control underwent different treatment processes, you may...the one shown in Figure 1.  Figure 3: An example of a histogram plot for a yes/no flow cytometry...

...are a few examples. Make sure you use a proper plasmid (i.e. contains the α-complementation cloning MCS...isopropyl β-D-1-thiogalactopyranoside). (To be completely accurate, IPTG binds and inactivates the lac ...to a lacZΔM15-mutant bacterial cell in trans complements the mutation allowing for a functional enzyme...enzyme. This process is called α-complementation. The system described above was put to practical use in the... the following way. Scientists engineered a multiple cloning site (MCS) into the α-peptide (represented... inserted it into a plasmid, creating an α-complementation cloning vector. When a cloning reaction goes...interrupted as shown in cell A, and thus will not complement the cell's β-galactosidase mutation. An unsuccessful...

...benefits gained by lowering the complexity of the screen. For example, the analysis becomes easier, the...discussed below. The software could also be used as a simple tool to rapidly find one or a few gRNAs suggested... the single cell level), are separated by, for example, cell sorting. The separated cells are subsequently...-defined, hypothesis driven research question. Examples of custom CRISPR screens Differentially expressed...a lasting impression on me. The slide showed a complex signaling pathway with many receptors, signaling...be very useful for drug discovery, to dissect a complex signaling pathway, and to identify what part of...potential components of a signaling pathway. For example, one could study the cellular response to a cytokine...

...researchers develop a variety of therapies. For example, many scientists use mice to study diseases such...such as cancer since mice better recapitulate the complex interactions between cancer cells, therapeutic ... in other model organisms or cell culture. For example, Scott Lowe’s lab utilized mice to study how different...Yeast (Saccharomyces cerevisiae) Yeast, one of the simplest eukaryotic organism, is a commonly used model ...in breads and other baked goods! Yeast is cheap, simple and easy to work with as they can survive in a ...diseases that are caused by a specific gene. For example, several genes mutated in human cancers have a ...recapitulate in vivo conditions similar to humans. For example, due to their simplicity, yeast have become an ...

...meant for isolating RNA from rat spleens in which they homogenized spleens in a guanidinium thiocyanate solution...tissues, cells, plants, fungi, or bacteria, keep samples cold and work quickly to mitigate RNA degradation...180°C for at least 4 hours.   If your final RNA sample(s) are resuspended in water or TE buffer, store...freezer.   RNA extraction methods evolved into a simple protocol still used today There are many alternative...for RNA extraction and purification. There is one simple method that works, and variations to that method...requires a lot of time and depending on how many samples you may have, access to one or more large, expensive...effectively separated from DNA and proteins by a simple extraction protocol with guanidinium thiocyanate-phenol-chloroform...

...nucleotides (for example, Digoxigenin-labelled Uracil) that will bind to complementary mRNA within an embryo...WISH protocol involves preparation of the tissue sample, several washes, and temperature changes to visualize...specific antibodies that detect the mRNA-riboprobe complex are added to the tissue. The last step involves...cells that contain this mRNA-riboprobe-antibody complex and, as a result, these cells turn blue. Students...organism of interest, use the RNA to create a complementary DNA (cDNA), and then use PCR and molecular cloning...cell types or entire organs in planarians. For example, the gene Piwi labels neoblast cells and the gene...one strand of the siRNA and are directed by complementarity to the remaining single stranded siRNA to bind...

...negotiation as something done not by people wearing a lab coat, but instead by people wearing business suits discussing...though three examples depend on many specific details about the individuals, each example provides a scenario...scenario where both people get something valuable. Importantly, as the third example shows, the ultimate... is important to people so that you can approach negotiations creatively with people from anywhere in ...transferable skill? Negotiation as a zero sum game Some people think about negotiation as a zero sum game - that...machine? Let’s walk through a more specific example. Perhaps you and your lab mate are negotiating ...your organization. In my non-academic roles, some examples of negotiation have been: A job offer conflicts...

...inexpensive and can be 5’-phosphorylated in house using a simple PNK reaction or can be purchased 5’-modified from...probably while it is being synthesized) can form a complex with an ssDNA guide. Therefore, researchers must..., the widespread success of CRISPR-Cas9 across multiple model organisms (3) lies in the versatility of...inject cells with in vitro preassembled Cas9/gRNA complexes or with guide RNA- and Cas9-expression plasmids...which could be less desirable if one has to target multiple sites in a single experiment. Also, the NGG PAM... an optimal distance from the target site. For example, NGG may not be present in AT-rich regions of the..., I was going through NgAgo google forums where people write their experiences with the new method and...

..., and conservation. But please be aware that there are many easy-to-implement confinement strategies that...with wild ones. For example, Drosophila lines with compound autosomes are completely infertile when mated...organisms with Cas9 risk creating a gene drive? The simplest way to make a gene drive is to insert an endonuclease... cell can create an RNA-guided gene drive. In principle, this event could happen even if there isn't any...authorities) was to detail these robust and easy-to-implement confinement strategies2. Our hope was that anyone...laboratory organisms but not wild organisms. For example, an sgRNA-only drive will spread exclusively through...with that sequence2. Both methods are easy to implement and have been tested in yeast5. Ecological confinement...

...I was careful to keep it relatively simple. I honed the complexity of the talk by running it by the teachers... Association (PTA) has been instrumental in supplementing that curriculum. It’s through the PTA that I...elementary students can ask. It doesn’t just need to be people who think of themselves as “scientists.” We had...for my school’s Math & Science Night. There are plenty of places to get ideas for science activities. ...Spangler Science The Fab Lab Middle school: supplementing the science curriculum Sharing my expertise ...will likely change for next time is giving fewer examples and think of a way to make this section more interactive...did not try to sugarcoat the potential dangers of people using the technology in an unethical manner, but...