...cleavage at 1-12 off-target sites; in contrast, SpCas9 may cleave at ~90 sites, according to Kim et al. ...in AT-rich genomes. Additionally, whereas Cas9 generates blunt ends 3 nt upstream of the PAM site, Cpf1... strength may lie in its low off-target editing rates, determined using sophisticated genome-wide analysis...many of its computationally predicted off-target sites, Cpf1 does not mediate detectable off-target cleavage...mechanism. To prevent new editing post-HDR, repair templates should remove the PAM sequence. Many research ...

...recommend no more than 40 minutes for the panel discussion leaving 20 minutes for audience questions (30...Follow Addgene on LinkedIn for repository news and updates. Panel discussions are becoming a ubiquitous format...members all say the same thing and one panel member dominates the discussion? Or maybe the one that goes way...interviews, the moderator should be taking good notes and discovering interesting stories, riffs or opinions...be good at talking, so they will easily take 5 minutes to introduce themselves and, voila, 1/2 of your...

...recombination using a repair template. These templates can include loxP sites to create conditional floxed alleles... targeting vector such that a set of three LoxP sites flank the resistance gene and the targeted exon ...replaced with the same sequence flanked by LoxP sites while the resistance cassette has been placed into...marker using Cre recombinase. Since there are 3 loxP sites, recombination can occur in multiple ways. The desired..., as seen in line 4 of Figure 4. Since the loxP sites are located in intronic regions, this gene will ...

...serotype AAV1. Distinguish dendritic neurites from axonal neurites with AAV vectors expressing cytoplasmic... the antibody toolbox, they screened antibody candidates using a synthetic human scFV library and characterized.... After finding no cross-reactivity, antibody candidates underwent affinity screening to select for slow...slow off-rate binders. The best candidates were validated by IP-MS using HEK293 cell lysate where the ...providing enzyme protection while still allowing substrates and products to diffuse freely into or out of...

...intracellular trafficking, and protein turnover rates. Additionally, by engineering FP fusions associated...individually generate gRNAs and homology arm repair templates containing the tag for each insertion. Also, it...digestion of the destination vector with SapI yields 2 sites- the first site accepts the sgRNA target sequence...be used to generate functionally unique repair templates. A final ligation reaction correctly assembles...genetic locus, the toolkit also offers repair templates for tagging proteins in a tissue specific manner... vectors for inserting a tag at multiple target sites. SapTrap vectors can be easily modified to tag hundreds...

...DNA into smaller pieces, and proximity ligation creates chimeric circles of DNA that are distant linearly...delivery of the two remaining CAPTURE 2.0 components eliminates the need to create stable cell lines and allows... beta globin locus. The two methods had similar rates of specificity for the DNA sequence targeted in ...interactions at once. Chromatin interactions at five sites in the well-characterized beta-globin locus were...negative control gRNA or predicted gRNA off-target sites. Long-range chromatin interactions identified with... a more vivid chromatin picture, CAPTURE 2.0 re-writes the script on gene regulation. References Han...

...to Microbial Media BitesizeBio: How to Get Better Plasmid Midiprep Yields BitesizeBio: Better Plasmid Midipreps...figure at right for an example). Look out for carbohydrates Certain strains, including HB101 and derivatives...derivatives, release large amounts of carbohydrates that can inhibit the cell lysis process. To ensure that...may lead to loss of the plasmid, and using older plates could increase plasmid loss or mutation. Let it...smaller cultures, especially those in deep-well plates, require a faster shaking speed for proper aeration...

...construct (i.e.,functional cassettes are recyclable) Can only work if the restriction sites used for the assembly...repeatability of the method. Note that the restriction sites of the MCS are only regenerated at the 5’ and 3’...free of specific restriction enzyme recognition sites. Thus, chaining methods are not necessarily suitable...sequences. Intermediate chaining products (called “cassettes”) can be readily reused in the assembly of any... et al., 1962), SalI, XhoI and MluI recognition sites, which contain CpG dinucleotides in their 6-mer ...

...an antigen binding site for a total of 2 binding sites per antibody. C) IgG’s are big enough that multiple...a double-edged sword. An antibody’s large size creates more distance between the target antigen and the...process can be avoided direct antibody fluorophore conjugates are used, but these are usually more expensive...varieties. This refers to the number of cysteine sites the nanobody has available for conjugation to a ...commercially available. References 1. Pleiner, T., Bates, M., & Görlich, D. (2017). A toolbox of anti–mouse... plasmids from this publication at Addgene. 2. Bates, M., Böhning, M.I., Chug, H., Görlich, D., Lee, ...

...Zuckerman, H. 1977. Scientific Elite: Nobel Laureates in the United States. Free Press. New York. Resources MDH...Knutson et al., 2010a,b).      Fig. 1: Undergraduates in Provost’s CURE prepare to analyze their ...the opportunity to make new discoveries. Provost notes the importance of acknowledging student apprehensions...original research experiences for thousands of undergraduates. The MCC develops and makes freely available... State College in New Hampshire. He mentors undergraduates conducting NIH- and NSF-funded research on ...

...lockdown. To allow our model to give uncertainty estimates for each parameter, we added log-normal noise ...only the 10 papers with more than 10 unique order dates in 2020. We fit our model for each region using ...Pytorch, which gave us the maximum likelihood estimates of each parameter. We approximated the data likelihood...rate of unique PIs/labs placing orders per day) on dates impacted by each effect. The inferred parameters...believe that 1/22/20–2/23/20 are the more accurate dates of lockdown in China, then the observed data is ...Medical Institute Investigator. Liu’s research integrates chemistry and evolution to illuminate biology...

...split-luciferase system, intein-mediated splicing reconstitutes functional luciferase only when fusions of the...for screening greater numbers, but the latter eliminates the step of identifying prey proteins after screening... can often be accounted for using appropriate replicates and controls (see Tips below), and by validating...two-hybrid screening Minimizing false positives: Run replicates of the experiment to reduce the likelihood of...high number of false negatives in Y2H screens. Testing your reporter system with a pair of proteins that...your fusion proteins physically blocks the binding sites for the protein partner or the UAS/reporter gene...

...activated” Cas13 produces a quantifiable signal that indicates the presence of your targeted nucleic acid. To...commercial lateral flow strips, similar to pregnancy tests. With lateral flow strips, the presence of your ...for using SHERLOCK to detect SARS-CoV-2 RNA. The test is started using RNA purified from patient samples...Cas12a however recognizes a different PAM site and generates 5’ and 3’ staggered ends after dsDNA breaks. The...per second. A quantifiable fluorescent signal designates the presence of your DNA of interest, in this...CRISPR-based platform on which an infinite number of tests” for biosensing can be built upon. Mammoth Biosciences...

...protein. Such flexibility makes aptamers ideal candidates for biosensors. Aptamer biosensors are used in...oligonucleotide-only sensor could save precious minutes in a time sensitive detection assay. In 1999, ... selection of Spinach with a target metabolite creates an aptamer that will only bind to DFHBI in the ...dependence. Though initially still unstable in eukaryotes, the Jaffrey lab has since developed new RNA-...RNA-based scaffolds to contain and stabilize Broccoli cassettes, thereby extending their use into mammalian cells.... The nature of aptamers makes them excellent candidates for developing assays for otherwise difficult-to-study...

...simultaneously. Image from Li et al., 2021.   The updates involved the following changes. In pCas9, we 1)...the ccdB gene containing BsaI restriction enzyme sites at its ends to generate pEcgRNA. The ccdB serves...32 hours instead of ~60 hours. We successfully tested the pEcCas/pEcgRNA system in 4 E. coli K-12 strains...strain, and 1 Tatumella citrea, with a total of 70 sites edited. We optimized the strategy for plasmid curing... into crRNA arrays targeting multiple different sites in E. coli. pCutamp cures the plasmids by targeting...Chinese Academy of Sciences. He joined Shanghai Institutes for Biological Sciences in 2000 and became a ...

... suitability for in vivo studies. Testing AAV-SaCas9 in vivo To test the efficiency of AAV-SaCas9 in vivo...Cas9 genome editing varies across targets, they tested two genes in mice. For both genes, they saw indel...polypeptides that, when at the ends of two proteins, mediates protein trans-splicing, similar to an intron in...proof-of-concept split intein SpCas9 which had modest editing rates in HEK-293T cells when compared to the full-length...genes in human cells using compact expression cassettes. Sci Rep 5: . https://doi.org/10.1038/srep10777...

...you realize events are plotted on an invisible Cartesian plane (right.) Each event is plotted by the amount...different population.  Gating and plot orders  As the gates progress from broadest markers to most specific,...conjugated to fluorescent markers are used to identify monocytes (upper right) and a mixed population of B cells...understand how the researchers decided to place their gates on the plots! Flow data is messy, so learning how... will have numbers by gated populations; this indicates the percentage of total events contained in the...intracellular IFNγ, IL-17 and IL-10 producing T cells correlates with the occurrence of post-transplant opportunistic...

...Creativity is required to develop new projects and test novel approaches, a necessary skill set for any ...be easy to optimize virus production with roller skates—though I could get around the lab faster that way..., easy goal. For example, set a timer for five minutes and come up with as many solutions as you can in... is simply to throw out ideas.  When your five minutes are up, if you haven’t come up with any ideas obviously... or even very frequent, but establishing a few minutes every month dedicated to thinking about the future...time crunch, your break can be as simple as a few minutes of meditation or a walk to the break room for a...

...sterile due to a failure of primary spermatocytes to enter meiosis. Testis of cookie monster mutant flies ...component of the fly actomyosin cytoskeleton that regulates cellular contraction. This network is composed...the formation of the visceral mesoderm which differentiates into the muscles of the gut. Embryos that lack...bagpipe: two homeo box genes that determine cell fates in the dorsal mesoderm of Drosophila." Genes & Development... "Myosin light chain–activating phosphorylation sites are required for oogenesis in Drosophila." The Journal...

...chalk talk." However, few, if any, faculty job candidates have seen an actual chalk talk. Their first exposure...session lasts up to an hour. You will get 5-10 minutes to present your research vision uninterrupted and...begin working through your aims over the next 45 minutes. The faculty will ask you lots of questions. Your...Comparing model A vs. B is more constructive than testing the validity of a single model. The first approach...is: What is the big question or mystery that motivates your research? This is the central idea. It should... Jennifer Lippincott-Schwartz at the National Institutes of Health, was an Associate Professor at Albert...