...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...

... 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...

...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 ...

... assortment toxins to claim territory or kill competing microorganisms, but did you know bacteria also...subunit of DNA gyrase and trapping it in the cleaved complex. This results in DNA breakage and cell death. Conversely...toxin-antitoxin roles. Restriction modification systems are composed of restriction endonucleases and methyltransferases...populations to retain more of these plasmids as compared to those without the restriction modification ...described, the hok/sok systems consist of three components: Host killing (hok), a long-living toxin that...K. Burley. "Crystal structure of the MazE/MazF complex: molecular bases of antidote-toxin recognition....

...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...

...across the entire blot. So how can you accurately compare different lanes? In relative quantification, the... conclusions, and it’s also problematic for computational quantification. Outside the linear detection...individual protein. This ensures you are truly comparing the abundance of your POI as a fraction of the...detection ranges of multiple targets. Convenient! Comparison of loading control approaches Cartoons are nice...all, but we’ll leave you with this real-world comparison of different loading control approaches from ...different methods of quantifying total protein are compared. While a higher slope indicates higher sensitivity...

...metabolites. Biotin acts as a carboxyl carrier from one compound to the other. These biotin dependent enzymes are...discovered in the 1960s, strepavidin. The Biotin-avidin complex can then be used and/or manipulated for a scientist...immunoprecipitation experiments. Biotinylated proteins or complexes can be easily purified using columns or beads ... sample. Any free biotin-binding sites on this complex can bind to your biotinylated antibody. This creates...creates a large meshwork of biotin-avidin complexes bound to your protein of interest leading to amplification...This strategy is referred to as the Avidin-Biotin Complex (ABC) immunohistochemistry (IHC). For more information...

... different universities are trying to compile more comprehensive demographic information about the postdoc...Postdoc: An Institutional Perspective. This lack of complete information does not, however, prevent us from...from the career panel was that there are many companies and organizations (including Addgene), that need...scientifically minded, and able to communicate complex information - all traits you are likely to find...others within their labs - as a sort of over-compensation, these mentors can treat the underrepresented...

... received his PhD in Computer Science from MIT. His research focused on computer systems and networks.... 4 - Developing Addgene as a modern e-commerce company and establishing credibility Chapter 5 - Realizing...Kenneth received his Bachelor of Science degree in Computer Engineering from Tufts University and his Master...and a Masters in Engineering from MIT, both in Computer Science.   Special thanks to our sound engineer...

...last year: The Christmas Decoration Competition! #ScienceFun #ZoomParty @Addgene pic.twitter.com/drNBUdlQco.... Nonetheless, Addgene’s annual Deck the Lab competition drew upon the creativity of the community, who...place-winning entry doubled as their lab’s decoration competition, and each lab member used an impressive variety...bacteria, fungi, and viruses. Final tree with the complete #MicrobeAdventCalendar ornament set (plus more...

...bacterial CRISPR engineering One common way of accomplishing bacterial genome engineering is done with recombineering...transposition. INTEGRATE consists of four major components including (1) a CRISPR RNA, (2) four proteins...transcription by physically blocking the elongation complex from binding the DNA or extending the transcript...transcription factors, phage proteins, and RNAP components (Dong et al., 2018). They found a transcription...transcription factor called SoxS for CRISPRa. Adding complexity, they found that they could also include a CRISPRi...systems is base editing. This process is generally accomplished by fusing an enzyme capable of converting one... targeting approach is then used to direct the complex to a specific locus. This approach provides a more...

...Drs. Koeris and Lu founded the biotech startup company, Novophage. As you'll hear Dr. Koeris explain however...Ventures, Koeris helped change Novophage's focus. The company went from developing therapeutics to developing...pathogens in food products. Along the way, the company changed its name from Novophage to Sample 6. To...using Sample 6 and why? 24:41 - 29:00 : Dr. Koeris compares and contrasts being a graduate student with being...personally take on the risk of founding a startup company? 32:20 - 34:37 : Whose responsibility is it to ...

...reporters Difficult to Multiplex: FRET reporters are comprised of two fluorescent proteins with very specific...encoded fluorescent kinase reporters. A KTR is comprised of a fluorescently-tagged substrate for a kinase...for kinase activity.  Advantages of KTRs When compared to FRET reporters for measuring kinase activity...of KTRs KTRs also have some disadvantages when compared to FRET reporters. First, KTRs monitor global ...measure kinase activity in specific subcellular compartments. Second, because KTRs shuttle between the nucleus...

...possibilities in the engineering of complex gene networks. A comparable system for targeting specific sequences... of RNA is highly desirable for extending the complexity of genetic circuits, allowing for tighter spatio-temporal... protein that binds an alternate sequence. By comparison, Abil explained, the specificity of other RNA..., designer PUF proteins can be fused to other components to lend them many other functions for use in ...

...markers. These are especially useful in protein complex studies where tagging of multiple protein products...for easy capture and detection of proteins and complexes, without the artifacts sometimes associated with...Formosa, at the University of Utah, has built a complete collection of yeast tagging modules with each ...ideal for tandem affinity purification of protein complexes. John Pringle and Jürg Bähler have deposited a...GST, or GFP). Longtine et al.(3) describe a complimentary set of plasmids for use in S. cerevisiae, with...

...The application process When I first saw the comprehensive job description for this co-op, I was immediately...description of the tasks I would be responsible for, in comparison to the other positions listed with a much less...training, I was gradually introduced to Addgene as a company and given more details on what my position entailed...they do and how it relates to other teams at the company. This provides new (and old) employees with information...Northwestern University. She is one of several students completing their co-op at Addgene. This is her second contribution...

...without the exo/polymerase generation of annealed complementary regions. Why use a high fidelity ligase? Numerous...the Ligase Detection Reaction (LDR), probes complementary to the sequence of interest are annealed to ...separate pools were prepared, each with a different complement strand covering all 16 possible NN pairs in the... system for direct identification of allelic composition of mutated DNA in a mixed population of excess...JS, et al. "A high-throughput assay for the comprehensive profiling of DNA ligase fidelity." Nucleic acids.... Pascal, John M., et al. "Human DNA ligase I completely encircles and partially unwinds nicked DNA." ...

...give you the reverse sequence, complement sequence, or reverse and complement sequence.  Filter DNA tool:...well as those deposited with Addgene. NCBI BLAST: Compares nucleotide or protein sequences to sequences in... tools help with that.  DNA sequence reverse complement tool: Enter a DNA sequence and the program will...DAD): This tool designs overhang sets for high complexity Golden Gate Reactions Cloning ligation reaction...