...Trends: Sequence based input is by far the most common with most tools also requiring that the user specify...CRISPR software embodies solutions that facilitate most experimental applications in CRISPR engineering....can invest our time into learning and extract the most value? This is only one of many decisions faced ...are two major areas where software can help. The foremost handles the design of sgRNA and represents the... suitability. Functions of this category are the most common between tools. Examples: “single-target design...design”, and “single-method design”. Trends: The most common utility functions are batch design or multiplex...detect off-targets. The few offline tools available mostly work on any computer but generally require working...

...is the chance of a chromosomal inversion, or just random mutations if the chromosome is repaired with all...small edits that change single base pairs, to chromosomal inversions and large deletions. Many of these...different types, with Cas3 systems making up type I, the most abundant type in the Class 1 category. These type...are largely underutilized in research settings — mostly a consequence of Cascade's complexity. Figure...an important component of the CRISPR Cas3 system. Most Cascade complexes are comprised of a combination...expression can still be a challenge. A multitasking Cas Most Cas effectors have one job — Cas see DNA, Cas chop...repeatedly cut the DNA at an efficient rate. These cuts most often occur in thymine-rich regions on the non-target...

...ligase is a NAD+-dependent DNA ligase from a thermostable bacterium that can survive high temperatures...advantage of the high fidelity of Taq and other thermostable DNA ligases. These applications detect specific...amplified fragment) and treated with a high fidelity thermostable DNA ligase. If the target sequence is present...helix shape (22). Large purine:purine mismatches and most smaller pyrimidine:pyrimidine mismatches are typically...several DNA ligases bound to substrate (23, 24). Thermostable DNA ligases, including Taq DNA ligase, are naturally...detectably ligate many T:G, T:T, and A:C mismatches. Thermostable DNA ligases are active at elevated temperatures...below). Conclusion It is important to note that thermostable, high fidelity, nick-selective DNA ligases like...

...two different pairs of chromosomes using gRNAs specific to sequences on chromosomes 9 and 13. They next ...example, the visualization of chromosomal translocations or chromosomal shattering (chromothripsis). As...catalytically dead Cas9 (dCas9) has many applications, the most well-known being transcriptional activation/repression...could then be used to examine both inter- and intrachromosomal dynamics, and how they change in response ...turned their attention to mapping pairs of intrachromosomal loci. The technique successfully resolved ... this work represents the first mapping of intrachromosomal loci, a major benchmark in characterizing ...next! References Multicolor CRISPR labeling of chromosomal loci in human cells. Ma H, Naseri A, Reyes-Gutierrez...

... biosensors for Human Milk Oligosaccharides (HMOs). HMOs are thought to play an important role in nuturing...to engineer bacteria for the efficient product of HMOs that could be used to supplement newborn diets and... cost-effective way to measure the production of HMOs. Current HPLC-MS and fluorophore-based technologies...biosensors that could quickly and reliably detect HMOs. Fatima’s first design is a whole cell biosensor...specifically proposes using this sensor to detect HMOs found on cellular surfaces. After incubating the...the sensor with bacteria engineered to produce HMOs, sensor binding results in an increase in fluorescence...fluorescence of cells that actively produce the HMOs. Fatima has demonstrated the successful production of both...

...when I recently attended the 2018 Chromosome Architecture and Chromosome Organization Keystone Symposium...nucleosomes, loops, chromosomal domains, or how stretches of DNA on different chromosomes interact with each...posters I saw at the 2018 Keystone Chromosome Architecture and Chromosome Organization. These aren’t the ...linear” is a misnomer. The DNA packed into our chromosomes is very much 3-dimensional, as you can see by...Shapes a Single Nucleus from a Set of Mitotic Chromosomes. Samwer M, Schneider MWG, Hoefler R, Schmalhorst...

...resistance cassettes is most commonly employed, with the KanMX being the most prevalent due to its versility...highly versatile DNA transformation system.Unlike most other microorganisms, yeast have both a stable haploid...and must be integrated directly into the host chromosome via homologous recombination. Yeast Replicating...Replicating Sequence (ARS) derived from the yeast chromosome. As the name suggests, these vectors can replicate...replicate independently of the yeast chromosome; however, they tend to be unstable and may be lost during...replicate as though they are small independent chromosomes and are thus typically found as a single copy...integration. Yeast Episomal plasmids (YEp): These are most similar to bacterial plasmids and are considered...

... but almost all of these guidelines can apply to any workplace or job. It amazes me when the most analytic...”  and hone in on the factors that will make the most difference for your success. Gather data on the ...if they are not local. Alumni meetings can be the most informative way of gathering data. Former lab members...totem pole). Can you see yourself enjoying this atmosphere? Lab size matters Yes, yes it’s all about ...the science but really it’s not. It’s about the atmosphere that will allow you to thrive. How big is the... the energy level in the lab? Does a frenetic atmosphere make you edgy or energized?   Are you an only...backbone”. The sad truth is, if it happened before, it almost certainly will also happen to you. As you speak...

...integrations on the genome. Most recently, we’ve developed a new system “Multicopy chromosomal integration using...bacteria. Our pCas/pTargetF system has been one of the most popular genomic editing systems in E. coli. However... both plasmids into the cell. The edit in the chromosome can now take place. After editing, pTargetF can...great for experiments that require only single chromosomal integration (ex: genome editing), our MUCICAT...transposase system, INTEGRATE, is best for multicopy chromosomal integration (ex: for protein expression) (Zhang...Zhang et al., 2020). Multicopy chromosomal integration for gene expression is an alternative versus plasmid-based...advantages: Fast and site-specific multicopy chromosomal integration of cargo without using screening ...

...There’s an astounding number of ways to create chromosomal mutations in bacteria, so many that it may be...quick and easy way to introduce a mutation in the chromosome is to disrupt expression of a gene with an antibiotic...resistance cassette. This leaves a “scar” in the chromosome, sometimes interfering with expression of surrounding...ones that don’t leave any undesired scars in the chromosome. These methods roughly fall into two categories...domesticated from the Lambda bacteriophage, is arguably the most widely and productively used  (Thomason et al., ...introduced from a conjugative plasmid replaces the chromosomal copy. This is a versatile technique, and is less... markers used to identify double crossovers? The most commonly used counter-selection system relies on...

...term familiar to almost every person regardless of profession, race, or gender. Almost no other disease...material is damaged beyond repair, that cell will most often commit itself to the apoptotic pathway and...eliminate cancerous cells from the body. One of the most prominent of these is the Ras pathway. The term ...exploiting the different mutant versions of Ras that are most prevalent in human cancers, and synthesizing compounds...mutated residues. For Ras researchers, one of the most exciting and relatively new resources is the RAS...Further, for each plasmid in the kit, the transcript most commonly found across all TCGA tumor samples is ...New resources and technologies become available almost daily, yet obstacles remain. One of the biggest...

...healthcare consultant, I have worked on most of these kinds of projects. Almost all of my projects, have involved...health insurance companies, and governments on the most effective and efficient ways of delivering healthcare...that specialize in business strategy tend to be mostly engaged by pharma and biotech clients. In this ...Who would enjoy healthcare consulting? One of the most rewarding parts of the job is being confronted with...healthcare space. The consultants who enjoy their jobs most, get pleasure from taking up such complex challenges...situations can get stressful and to help manage that, most consulting firms coach and counsel employees on ...of the day, the consultants who enjoy their jobs most are those who understand that these challenges are...

...seven most popular serotypes available from Addgene from 2016–2023. B) AAV preps for the seven most popular...quickly became one of our most popular ready-to-use viral vectors, making up almost 10% of our requests by...vector tools, which may help explain why they are the most commonly requested (Figure 3). Over 400 biosensors...containing a genetic payload. While AAV9 and AAV1 are the most requested serotypes (Figure 4), our catalog includes...action potentials that GCaMP6 and jGCaMP7 do not. Most likely, it’s a combination of many factors unique...significant insight into which vectors are likely to be of most use to the community. Thoughtfully prioritizing ...

...of recombinant protein expression and some of the most popular commercial strains designed to avoid them...usage (Table 1) A few mutations are common to all or most expression strains to accommodate high protein levels...  The DE3 lysogen/T7 promoter combination is the most popular induction system. The DE3 lysogen expresses.... Although the DE3/T7 RNAP system works well for most experiments, the lac promoter can “leak,” meaning...exists even without the addition of IPTG. This is mostly a problem for toxic protein products, which can...should generate sufficient expression levels for most purposes, but what do you do when you’ve tried a...solubility concerns. Insoluble and Secreted Proteins: The most common purification protocols are designed for soluble...

...making it unsuitable for most in vivo applications.  Third, even in the most optimized tissue-culture ...sequence-specific knockins - for which the AAV approach is most well suited, since it routinely generates knockin...this is comparable or better than the efficiency of most current CRISPR-based approaches for the generation...several recent reviews (Howes and Schofield, 2015; Moser and Hirsch, 2016; Epstein and Schaffer, 2017) Hopefully...Jung-Sik, et al. "Intact cohesion, anaphase, and chromosome segregation in human cells harboring tumor-derived...engineering using adeno-associated virus (AAV)." Chromosomal Mutagenesis. Humana Press, New York, NY, 2015...2015. 75-103. PubMed PMID: 25408402. 9. J Moser, Rachel, and Matthew L Hirsch. "AAV vectorization of DSB-mediated...

...still not get the most from your team. It is pretty clear that if people are mostly happy at work, they... and grow in skills. Managing a team is hard and most people have some learning they need to do before...overwhelmingly agree that these are the factors that most contribute to job satisfaction: A feeling of being... not mean a manager must allow his team to work mostly at home. Having a high degree of freedom and diversity...pursue and problems to solve (I think this is true of most people, not just scientists) Are self-directed, ...be in touch with the needs of the team and to be most effective in their role. The best managers have ...

...this useful. Now that we’ve been doing this for almost 6 months, you may be looking for more ways to make...don’t all get the mentoring support we need, and most of us could use more mentors to listen, guide and... virtual!   2. Practice public speaking For almost any science career, there will be a time when you... about trying your hand at a video presentation? Most video platforms make it easy to record yourself....audiences in your future.     3. Start something In almost every career talk I present, I’m asked what Addgene... different criteria for different roles but for almost every position, we look for candidates that have... interviews, for more details on how to make the most of these exploratory conversations. If you set yourself...

...sites on different chromosomes in living cells, the system requires high-copy chromosome-specific loci in...study the spatial and temporal arrangement of chromosomes and how these arrangements may affect nuclear...based multicolor labeling system to visualize chromosomal loci in human cells (Ma et al., 2015). To do ...inter-locus distances. They tested this system out on chromosome 19 by targeting 46 repetitive loci that contained... (PR1), the p-arm (T1), and the q-arm (T2) of chromosome 19. Image from Ma et al., 2018 with permission...Hanhui et al. “Multicolor CRISPR labeling of chromosomal loci in human cells.” Proceedings of the National...

...the emission of photons. The most familiar bioluminescent organism for most people is the firefly (Photinus...pyralis) and perhaps not surprisingly it is also the most commonly used bioluminescent reporter. This beetle...from the firefly beetle (Photinus pyralis) is the most frequently used bioluminescent reporter, other luciferases...substrate coelenterazine and is likely the second most commonly used luciferase. Other organisms from which...assay The typical luciferase-containing plasmid is most often used to investigate the effect of regulatory...Many luciferase assays require cell lysis as the most efficient means to disrupt the cell membrane and...

...which chromosome the transgene has integrated and make genetic crosses with balancer chromosomes to make...that the transgenes can be inherited on the same chromosome. Targeted Insertion allows for direct comparisons...longer because you need to determine in which chromosome your transgene has integrated. This can be accomplished...accomplished with a few matings to balancer chromosome lines but is not necessary with targeted insertions...insertions (here you would know beforehand on which chromosome your docking site is located). This amounts to...genomic locus of integration is. The white gene is most often used, but other selection markers include ...