...-target effects and/or toxicity, it would be advantageous to instead use a preexisting, conserved cellular... just that as part of their CRISPR work in rice plants. Yang was intrigued by the tRNA processing machinery...gRNA expression. Since tRNAs are among the most abundant molecules in a cell, this system is clearly also...leading nucleotide could be processed, a distinct advantage over other methods that favor certain nucleotides...also examined editing efficiency in intact rice plants, comparing traditional gRNA constructs with PTGs... five gRNAs were tested, they found that 50% of plants had biallelic mutations at all five loci. This ...potential of PTGs. Applications of PTGs The major advantage of the PTG system is its universality; robust ...

...spontaneously occuring resistant mutants and the insensitivity of yeast strains to some antibiotics. Auxotrophy ...complex internal cell structures similar to those of plants and animals. Unlike bacteria, yeast can post-translationally...yet they still share many of the same technical advantages that come with working with prokaryotes. This...limited to: rapid growth, ease of replica plating and mutant isolation, a well-defined genetic system, and a...yeast cells, in addition to the bacterial ORI and antibiotic selection markers.  The yeast origin of replication...have utilized auxotrophic selection rather than antibiotic selection when working with yeast, due to high...higher copy numbers. Additionally, improvements in antibiotic selection have made utilizing the more traditional...

...uncover new gene variants associated with conditions of interest, then studying those variants in model systems... differentiate those mutant and wild type stem cells into any cell type relevant to the disease in question...efficiently generate human stem cells edited to carry mutant versions of 15 different disease-associated genes...together, there are occasional errors. That can be advantageous if you are trying to knock out a gene. One can...homology-directed repair by flooding the cell with a mutant template and fooling the cell into using that template...implicated in disease or, in cases where particular variants or mutations have been reported in people, we ...The TALEN kit is quite useful in generating the mutant cell lines that one needs to do disease modeling...

...amount of siRNA to be delivered or anticipating this response are important considerations when using these... how these structures are generated, why it’s important for cells to sense their formation, and how clinicians...This pathway has been clinically exploited for antiviral purposes similar to that of vaccines. If you know...is certainly a double-edged sword – balancing antiviral benefits with the inflammatory response associated...RNA hybrids. Unlike dsRNA, DNA:RNA hybrids are abundant in normal cells and are arise from a ssRNA engaging...However, transcription of thousands of genes is constantly occurring throughout a single cell, so these ...in turn lead to genome instability. Thus, it’s important to strike a balance between productive levels ...

...requirement for quantitative live-cell imaging, it is important to be able to quantify the photostability... non-linear dependence on excitation power is important since different fluorescence imaging strategies...Selecting a photostable protein The issues with quantification of photobleaching rates can be summarized as...water/oil emulsion at high power, it is far more relevant to measure how fluorescence intensity evolves ...higher excitation power, the photostability of the variants is comparable (lower panel of figure 1). To avoid...avoid changes in excitation power, it is important to perform the experiments on a single day on a stable... exchanging excitation filters). This avoids substantial changes in excitation power and will help to ...

...induced and quantified by measuring the amount of radioactivity in the culture supernatant (this is indicative...relatively pH-insensitive RFP variant, DsRed.T3 and a pH-sensitive GFP variant, super ecliptic pHluorin (... inhibitor. While this method does provide a quantitative measure of autophagy, it only measures bulk ... the protein. To monitor autophagy, the Pho8Δ60 mutant protein is expressed. Pho8Δ60 localizes to the ...final read out of this assay. This is also a quantitative measure of autophagy, but, like the protein ...sequence. Under normal growth conditions, both variants are excluded from the vacuole and emit red and...the cytosol-targeted and mitochondria-targeted variants are available from Addgene. References 1. Rosado...

...into research. Why are sex and gender analysis important? Doing research wrong costs lives and money. According... goal of gendered innovations It is crucially important to get the research right from the beginning. ... one of them. Why might the sex of the cell be relevant? Research shows that there are sex differences... and arbitrary decision. Unfortunately, it also meant that, by the very nature of the way the experiments...unique to male stem cells. Nor did they detect important differences in function between male and female...results, but this result shouldn’t be taken for granted for all research going forward. It’s just not that...many factors that interact with sex, and it’s important to analyze these factors during an experiment....

...mutations, antibiotic resistance, restriction sites used during cloning, and most importantly, the predicted...any other important features that differentiate this plasmid from its predecessor. Important features ...organize your own plasmid collection, although you will want to keep digital copies of your predicted sequence...sequence verify the plasmids. This is the most important step in ensuring quality, as it will catch any...sometimes have to design new primers to sequence the important features. You can use primer design software such...your primer binding site and the first feature you want to verify because you won’t get reliable sequence... amino acids worth, so make sure the feature you want to verify is within this range. Read some of our...

... a mouse model, and its success is not always guaranteed. To learn more about this process check out our... embryos as Cas9 from S. pyogenes and has the advantage of being smaller (Zhang et al., 2016). For editing...components can be electroporated into the oviduct of pregnant females, a technique called Genome editing via...injection into either an oocyte or into the oviduct of pregnant females (Yoon et al., 2018, Mizuno et al., 2018...replacement of the murine sequence with a human gene. Advantages of using CRISPR for mouse genome engineering ...the efficiency discussed above extends 4 major advantages of CRISPR mouse editing when compared to more...is much more efficient at generating the desired mutant offspring, with 85% success compared to 50% when...

...the gene is constitutively-active or a dominant-negative mutant. Reducing the copy number can also mitigate... gene repair with oligodeoxynucleotides: wanted and unwanted target locus modifications.,” Mol. Ther.,...useful for genetic engineering approaches. Most importantly for this technology, however, is that the transposase...of interest. The piggyBac™ system allows for significantly enhanced random integration efficiencies that...should be tested in the cell line of interest. Importantly, by varying these ratios, you can either maximize... If the cell’s chromatin structure undergoes significant rearrangement (such as when a stem cell initiates...transposon sequences.  The use of PBx is particularly important to avoid any possible random re-integration from...

...genome editing in plants While there have been many Cas nucleases developed for plants, it has been difficult...Cas-mediated genome engineering in plants. Read the paper in bioRxiv Find the plant genome editing toolkit ...the Cas14 plasmids Anti-CRISPR proteins for optogenetic control of CRISPR-Cas9 Anti-CRISPR proteins that...specificity of multiple wild-type and engineered variants of Cas nucleases. In doing so, they also created...discovered in 2017. Now Dominik Niopek’s lab has adapted anti-CRISPR proteins for optogenetic control of CRISPR-Cas9...post Read the paper in Nature Methods Find the anti-CRISPR plasmids Tagging human transcription factors...

...Cas protein variants may also be useful for targeting ncRNAs. For example, the Cas12a variant can cleave...RNAs in Plants with the CRISPR-Cas Technology is a Challenge yet Worth Accepting. Front Plant Sci. 2015...transcribed but do not encode proteins. They play important roles in many cellular and regulatory processes...into the break sites. Knock-in experiments are important for visualizing ncRNA localization and function...complex and not yet fully understood, so it is important to carefully select the target locations of knock-in... ncRNAs.  CRISPRi and CRISPRa For scientists who want to modulate ncRNA expression without permanently...genomic elements (Hazan and Bester 2021). Other Cas variants Another option is CRISPR/Cas13, which targets ...

...nucleoporin. Lu et al. Plant J. 2010 Yuhai Cui Red mScarlet Split-wrmScarlet variant of mScarlet Split-wrmScarlet...Complementation (BiFC). It can provide a qualitative or quantitative readout of the interaction in vivo or in vitro...includes I152L in N-terminal fragment An enhanced recombinant amino-terminal acetylation system and novel in...aa 155), includes I152L in N-terminal fragment Constant rate of p53 tetramerization in response to DNA...-10) and sfCherry2(11), also photo-activatable variant PAsfCherry2(1-10) Improved split fluorescent proteins...presence of heme oxygenase for chromophore formation (abundant in eukaryotes) A Near-Infrared BiFC Reporter for...presence of heme oxygenase for chromophore formation (abundant in eukaryotes) Bright monomeric near-infrared ...

...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 ...part of a study that examined the repertoire of antibodies from 19 COVID-19 convalescent individuals (Tong... 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... COVID-19, aside from the widely used qPCR and antigen tests.  Labs including Jennifer Doudna’s lab and...

...portable quantification of diagnostic results.  Our approach now detects samples at clinically relevant concentrations...in a clinically relevant setting. Molecular diagnostics for the Zika virus predominantly look at viral ..., and North America. Diagnostics will play an important role in helping to monitor and slow this spread... synthetic gene networks for the detection of antibiotic resistance and strain-specific diagnostics for...develop our paper-based platform into clinically relevant diagnostics for the Zika virus was daunting. It...American-lineages). Strain discrimination is especially important in the current outbreak because of the neurological...Pardee and Alexander Green! Keith Pardee is an Assistant professor in the Leslie Dan Faculty of Pharmacy...

...applications. As individual libraries can vary significantly, it’s important that we have an amplification protocol...and the desired coverage for the library (How important is it that every single plasmid in your library...protocol. Created with BioRender.com.   The most important factor to consider is your desired library coverage... plasmids in your library. As an example, if you want 100x coverage of your library, and your library ...500,000 colonies.  Pro tip! Since you probably do not want to individually count 500,000 colonies, you’ll likely... is less than that of electroporation, but an advantage is that chemical transformations are generally...overtake larger plasmids, if the library varies significantly in plasmid size). On plates, each transformed...

...he didn’t want to spend hours reviewing papers to piece together the best protocol;  he wanted to get going...how to prioritize but I  just added the “most important” tasks my schedule. This was bad for 2 reasons...to focus on what you should be doing : writing a grant, thinking about the next project, reaching out to...expectations, answer any questions, and (most importantly) decide on a timeline. My experience is that ...your team’s success depend on it.  Equally as important is to be able to actively listen - this can be...working with X?”  is going to get you what you really want to know. Remember that one on one discussion can...counter-productive. I always assume that reports want to succeed at work and providing feedback is needed...

...glass cloning rings to isolate colonies of antibiotic-resistant cells; during the time required to grease... video below) is simple: After obtaining antibiotic-resistant colonies, use a Sharpie to circle 12-48 ... do not normally use antibiotics. Lastly, and perhaps obviously, it is important to have a functional ... inside of the wrist or the cheek. (Parents of infants will have no problem with this!) Make sure your...levels of expression of our gene of interest. Advantages of the agarose cloning method The agarose technique...cells did not seem to form discrete colonies but constantly migrated away from each other. In our experience...morphology or other physiological properties. An advantage of this technique is the potentially large number...

... the participants had certainly realized the importance of seeking out mentoring as an important aspect...Kind of Mentor Do I Want To Be?” In this excellent essay Acclimatrix, an assistant professor at a research...current mentors/advisors like Yoda? If so, you might want to find additional sources of wisdom. While somewhat...thoughtful treatise on the kind of advisor/mentor she wants to be for her new lab. If only every science trainee...dedicated person guiding the start of their career. “I want to be a PI that holds my students accountable for...being coddling, to listen without being taken advantage of, and to respect my students as developing colleagues... If you have a question about mentoring that you want us to cover, let us know.   ...

...skills. Why are transferable skills so important? It is important to identify which transferable skills...means you’re guaranteed to get the job, however, many times the hiring team will also want to examine not...of demanding courses, a capstone paper I really wanted to hit out of the park, part time work, bills, ...through school, jobs, and volunteer work, and I guarantee you have developed them through similar experiences...potential employers, collaborators, or co-workers want to know that you are a well-rounded individual with...skills, before offering someone their dream job; They want to know if the candidate has what it takes to thrive...ask about your creativity, your potential manager wants to hear a specific time that you thought outside...