...specific chromosome. This image represents the composite of fluorescent measurements from red, blue, and...modified GFP-dCas9 and the gRNA scaffold to enhance complex assembly, thereby decreasing the amount of background... labeling efficiency was similar to that of a comparable FISH method. Importantly, they did not observe...with the previously established physical map. In comparing pairs of targets ~2 Mbp apart, they noticed that...that they could evaluate the degree of chromatin compaction even for this small distance! To the authors’...CRISPRainbow generates 6 unique colors and a white composite. gRNAs with two identical hairpin binding domains...

...significant amount of flexibility compared to a strict single-compatibility sequence like NGG. Nonetheless...NG, a rationally engineered Cas9 with NG PAM compatibility, as the name implies (Nishimasu et. al, 2018...know and love about Cas9, but with broader PAM compatibility. This alternative PAM requirement adds a significant...PAMs and is capable of editing at frequencies comparable to SpCas9. SpRY can also edit NYN PAMs, but with... Ai C, Hu J. In-depth assessment of the PAM compatibility and editing activities of Cas9 variants. Nucleic...

...blood clotting, and as we all know, a structural component of bones and teeth. It is an enzyme co-factor,..., a neuron subtype) and specific subcellular compartments for measurements. All of the sensors discussed...calcium concentrations in different subcellular compartments can vary greatly. Additionally, the calcium ...susceptible to artifacts and pH interference, and comparison between experiments can be more difficult due...GCaMP6 only) or mammalian cell expression, and compatible with constitutive or Cre-dependent expression...Genetically encoded calcium indicators to probe complex brain circuit dynamics in vivo. Neuroscience Research...

...science and collaboration. The field already has comprehensive reference pages and curated databases for scientists... (CeNGEN) The CeNGEN goal is to establish a comprehensive gene expression atlas of every C.elegans  neuron...that is creating the “first virtual organism in a computer.” This project has produced various resources ...four-dimensional contrast interference (DIC) microscopy and computer imaging processing. The combination of these methods...methods provides unique opportunities to develop computational methods to better understand animal development...Hammarlund, Marc, et al. "The CeNGEN Project: The Complete Gene Expression Map of an Entire Nervous System...

...remodeling. Chromatin is a complex of DNA and histone proteins that compacts cellular DNA into chromosomes...in the compacted region and results in a loss of gene expression. In this way, genes in compacted regions...and tightly bound DNA is to histones, the more compacted it becomes. This prevents the cellular machinery...have huge impacts in the way we study and treat complex or multifactorial diseases, enabling faster diagnosis...

...know that this process only became even more complicated with COVID-19 and standards for hybrid, virtual...without the financial and logistical stress that accompanies traveling for campus visits. This is definitely...where the PhD application cycle becomes much more complex. “How do I reach out to potential mentors?” “Where...very valid questions, and often make an already complicated process even more difficult. I’ve learned that...to be incredibly useful, as it gives a more comprehensive idea of that advisor’s research interests which... for the interview. Having notes close to your computer with things you want to highlight in the interview...

...while also ensuring that my work gets completed? I want my company to pay for me to attend a conference... How can I negotiate my starting date with the company so they know I’m excited while still being able...want to learn more about a different area of the company. How can I negotiate with my manager to gain experience...conference. How can I negotiate to show the company that investing in me is also beneficial to them? I have an...but I need help from another person/team at the company. How do I negotiate with the person/team for help...

...bacterial strains, like NEB Stable. NEB Stable competent cells lack RecA, a protein that aids in the homologous...helps me figure out if I have enough plasmid to complete my experiment. AAV is usually the limiting reagent...produce more virus because I don’t have enough AAV. Compared to the Addgene AAV Production protocol, I take...concentrate the purified virus. This process can be completed in one long day, or the virus can be stored at...set can be used and samples are quantified by comparing to an AAV standard curve, which is generated from... plasmid. This whole process takes ~3 hours to complete: 1 hours hands-on time and 2 hours for the qPCR...

...require a single-component nuclease to mediate cleavage rather than the multi-subunit complex employed by ...repair templates should remove the PAM sequence. Comparing Cpf1 and Cas9 on-target and off-target efficiency...lower than that of the widely used SpCas9 and comparable to SaCas9. As seen with Cas9 orthologs, Cpf1 ...sophisticated genome-wide analysis. At many of its computationally predicted off-target sites, Cpf1 does not mediate...sites, according to Kim et al. Kim et al. also compared the ratio of total off-target to on-target modification...

...These insights are hard to appreciate without computational support. Indeed, rational sgRNA design is not...experimental applications in CRISPR engineering. When compared to that collective utility, it’s easy to fault...tool unique. The CRISPR Software Matchmaker is composed of these features and enables you to select the... based on their comfort level with operating a computer.  Examples: “offline”, “online”, “CLI”, and “GUI...few offline tools available mostly work on any computer but generally require working knowledge of the... power of each tool they are interested in by comparing their past results to the designs suggested by...pattern at any edit distance. It won’t, however, compute any score other than the edit distance. Added functionality...

...sites compared to PE1. The pegRNA: A template and guide all in one The other important component of prime...favor copying the edit onto the unedited strand, completing the process. Let’s examine the parts in more ...Cas9 and reverse transcriptase To decrease the components prime editing would introduce into the cell, ...in higher editing efficiency. They called this complex PE1. Building upon prior reverse transcriptase ...homology to the genomic DNA locus. Sequence complementary to the nicked genomic DNA strand serves as a...

...division. These non-plasmid-containing bacteria can compete with and confound the goal of your experiment —...spectinomycin is the more stable and expensive antibiotic compared to streptomycin. Spectinomycin/streptomycin can...option! Otherwise, since it isn’t universally compatible with all bacteria used in research, it isn’t ...express the Tn5 element, but thankfully, most competent cells do not express this factor. Zeocin is handy...host/plasmid DNA during selection if it’s not completely bound up. While this likely occurs at a low rate...Stability Time Saving?  Works for multiple organisms? Compatible with most bacteria? No satellite colonies? ...

...immune system; and reconfiguring of the nuclear compartment, gene expression, and protein translation. Its...producer cells where there is E1 and E3 gene complementation. In target cells that are infected by the vector...virions) are not expressed. Note that replication-competent AdV vectors also exist and are used in certain...topic, which is that natural AdVs, replication-competent viruses with a recombinant genome, and adenoviral...higher than other viral vectors (tens of kilobases; compared to only 4.7 kb for AAV, 9 kb for retroviruses)...Tales: From the Cell Surface to the Nuclear Pore Complex. PLoS Pathog, 11(6), e1004821. https://doi.org/...

...Sharing data and community concerns AAV vectors are complex biological reagents; many factors, like storage...literature, scientists can use the Data Hub to compare different methods and vectors, and use this data...through word of mouth or old lab notebooks and computer files. If you asked your team members or colleagues...much unpublished AAV data do you have sitting in computers and notebooks right now?, I’m willing to bet the... rat CNS: AAV PHP.EB vector dose–response and comparison to AAV PHP.B. Gene Therapy, 25(5), 392–400. https..., C., & Flotte, T. R. (2015). Stability and compatibility of recombinant adeno-associated virus under ...

...just one or two recombination events, homemade competent cells of 107 CFU/μg are perfectly fine, yet performing...performing multiple modifications will require ultracompetent (109 CFU/μg) cells. Primer design Using specific...insertions, deletions, and point mutagenesis to complex assembly of multiple genes. Primer design for each...the forward primer by adding a short sequence complementary to the 5’ end of the reverse primer. Mutagenesis...efficiency of assembly decreases as the cloning complexity increases. The efficiency of recombination is...increased with greater homology, so performing more complex procedures can be aided by extending homologous...

... and/or are too expensive, time-consuming, and complex to use outside of standard laboratories. In the...step allowing for greater quantitation without compromising sensitivity. To detect multiple targets in ...signal of a single target. Csm6 can cleave ssRNA complementary to a crRNA of interest (Niewoehner and Jinek...with high sensitivity and specificity without compromising speed, ease of use, and portability. This method...molecular and clinical diagnostic applications. The company Mammoth Biosciences has been started based on DETECTR...ES, Koonin EV, Zhang F (2016) C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector...

...find a comparable library if it’s gone stale or we will need to update our code to be compatible with the...parts to our software, which has grown with the company. It would be easy to assign each developer to a.... Third party libraries are reusable software components that can be incorporated into software. You can...or they make significant changes that make it incompatible with our software. When this happens, we need...took us a total of two years, from planning to completion, to do this upgrade while continuing to add new...

...morpholinos. When injected into zygotes, morpholinos complementary to a target mRNA inhibit its translation either...observation unlocked novel research into genetic compensation, a previously unknown mechanism of gene regulation...established body plan including head, eyes, and tail. Compared to the opaque mouse embryo, where similar processes... been tracked using microscopy and advanced computational approaches (Keller et al., 2008). This microscopy-based...generation time and labor-intensive maintenance compared to invertebrate model systems, and their resistance...References El-Brolosy, Mohamed A., et al. "Genetic compensation triggered by mutant mRNA degradation." Nature...

...editing is the potential for off-target effects. To compare the off-target effects of SpCas9 and SaCas9, Zhang...normal and liver injury markers were not increased compared to a control AAV-GFP. Not only did the AAV-SaCas9...had modest editing rates in HEK-293T cells when compared to the full-length SpCas9. In 2016, Chew et al...cleavage of HBB and CCR5 genes in human cells using compact expression cassettes. Sci Rep 5: . https://doi....Desmarais J, Nogales E, Doudna JA (2019) CasX enzymes comprise a distinct family of RNA-guided genome editors..., Nureki O (2014) Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA. Cell 156:935–949...

...research proposal will distinguish between competing models. Comparing model A vs. B is more constructive than...about the time and resources required to feasibly accomplish the aims and clarify what type of aim you are...scaffolds with patient stem cells to generate compatible functional livers.” Why are you the best person...exist to solve the problem. What will you have accomplished in 5-10 years? You need to be able to describe...learned and/or now be possible to do? Research accomplishments are remembered for the problem solved, not ...