... Validating knockout mutations When a knockout cell line is desired, any mutation that introduces a frameshift...silent mutation in a neighboring nucleotide along with your desired edit. This silent mutation should ...cells were appropriately edited for your desired mutation - insertion, deletion, or site-directed knock-...is. If you are trying to introduce a homozygous mutation, it’s helpful to know how many copies you are ...Secondly, categorize your edit – is it any frameshift mutation? A specific insertion or deletion? A knock-in?...large deletion events.   Validating knock-in mutations You’ve introduced a donor template along with ... editor. What next? When generating a knock-in mutation, the best screening method depends on what your...

...in Figure 3. Though Cas9 is not a static molecule in any binding state, RNA loading stabilizes the closed...pyogenes Cas9 in the apo state (PDB ID 4CMP, top) compared to the guide RNA-bound state (PDB ID 4ZT0, bottom...Crystal structure of S. pyogenes Cas9 in the apo state (PDB ID 4CMP) with domains colored as in Figure ..., 2018), but it’s most often shown in an “open” state, with the NUC and REC lobes laying open like a book... that recognizes the PAM is so flexible in this state that it doesn’t appear in the crystal structure ...very weakly, most likely through unstructured electrostatic interactions. It also can’t recognize PAMs, ...region of the CTD, which is disordered in the apo state and absent from that structure. HNH, red; RuvC, ...

...unfavorable donor-acceptor orientation limiting FRET efficiency, circular permutation (Baird et al., 1999) ...Resonance Energy Transfer. Unlike the typical excitation and emission of an excited fluorophore, FRET ...typical steps in FRET are: 1. Donor fluorophore excitation by absorption of a photon 2. Energy transfer ... of the acceptor fluorophore back to its ground state by emission of a photon with wavelength specific...spectrum of the donor fluorophore overlaps with the excitation spectrum of the acceptor fluorophore. Otherwise...correlates with a change in the distance and/or orientation of the FRET pair. Since many biological processes...noise to the measurement. If the distance or orientation of the donor and acceptor proteins is not optimal...

...rely on that clone not having any confounding mutations, the risk is high. You are generating a gene therapy...patients and the diseases associated with an elevated mutation burden. The CRISPR platform must be optimized ...off-target sites. For example, if there are 10 putative off-target locations you can 1) determine if editing...hardly any flexibility in gRNA placement (e.g., mutating a specific amino acid or introducing a specified...evoCas9 are a few spCas9s which have been evolved or mutated to be more specific for exact gRNA matches (Rabinowitz...occurs. 2 gRNA approach Off-targets that come from mutations arise from base/prime editing or Cas cleavage....cleavage. In the case of Cas cleavage, off-target mutations can be mitigated by using two gRNAs in close proximity...

...balanced against the limitations of the assay.      Fig. 1: A schematic representation of Sybr Green ...PCR-based applications (qPCR and ddPCR.) qPCR Quantitative/real-time PCR (qPCR) has been used for copy ...amplicons under 100 bp (Ma, 2015.) A schematic representation of the Taqman reaction can be seen to the left...each reaction, math happens (this time, Poisson statistics), and the readout gives an absolute value of ....  Whichever method you choose, resist the temptation to over interpret your results: Sybr cannot be...product on a gel. And only ddPCR provides absolute quantitative data, instead of relative quantification.  Even...Arya N, Patel HR. Basic principles of real-time quantitative PCR. Expert Rev Mol Diagn. 2005 Mar;5(2):209...

...outlined below (or emission wavelenght range).  By mutating GFP, the variants blue FP (BFP), cyan FP (CFP)...breakdown of GFP, it's variants, and their relevant mutations, check out Marcy's post from last week on GFP....switch their color when activated by a specific excitation wavelength. This means that the emission wavelength...wavelength can change. In a few cases, the initial state of the protein is non-fluorescent, thus allowing...George G. Stokes) is the shift in wavelength from excitation to emission. For most FPs, Stokes shift is less.... Fluorescent Sensors: These FPs change their excitation/emission behavior upon environmental changes ...also is also called Bimolecular Fluorescence Complementation (BiFC). 8 points to keep in mind when choosing...

...CamR) by introducing a point mutation (H193Y). The reversion of this mutation by an adenine base editor ... Fulbright Visiting Scholar at the Pennsylvania State University. Imagine you are dealing with a defective...relies on the efficient delivery of donor DNA to facilitate repair. Since the development of CRISPR-mediated... of different basic BE technologies and their adaptation for plant genome editing. Cytosine base editors...desired T-A base pair. Figure 1: Transition mutation (C>T, G>A, A>G, and T>C) installation by base ...systems. Thus, the Yang Lab at The Pennsylvania State University (PSU) is adapting and improving base ...base editor (ABE) vectors to install A>G or T>C mutations in the plant genome. We constructed three versions...

...photostable. It also has narrow excitation and emission peaks (max excitation at 493 nm and max emission at...Scintillon Institute, Allele Biotechnology, Florida State University, Indiana University, and the Karolinska...than the most commonly used GFPs and YFPs. Its excitation maxima is at 506 nm and its emission maxima is...fluorescent proteins and therefore can be used in immunoprecipitation experiments with other fluorescent proteins...variants, including some potential tetramer-breaking mutations. The library was then screened through successive... replacements relative to CFP484. Because the excitation and emission maxima were red-shifted (462 nm ...researchers recommend using mTFP1 with a 445/30 nm excitation filter, a 470 nm beamsplitter, and a 495/30 nm...

...reliable feature annotations. Even with these extensive efforts, such an algorithm has limitations. For instance...database. Another limitation is that by tolerating mismatches, our algorithm could annotate a feature inaccurately...SnapGene’s most popular aspects - its ability to annotate a raw plasmid sequence and display frequently ...boundaries are less obvious. We found that the annotated boundaries for control elements in commercial ...the database – we will work to fill any common annotation gaps in the plasmids available through the Addgene...Collections so that a group or organization can annotate their sequences from a shared database of custom...features. Second, customers have asked for a way to annotate the genomes of newly sequenced bacterial or viral...

...and your presentations engaging and memorable. If you read my story above about the presentation faux pas...these things naturally than to give a dynamite presentation?  To that end, let’s chat about giving science...guidelines will apply to any type of science presentation, but, for more detail on the job talk for an...not talking about lab meeting here. Lifeless presentation of experimental results in the order they were...were performed does not make for a memorable presentation anywhere but in a lab meeting (maybe not there...there either). Think Keystone Keynote presentation or Ted Talk. You want to give the audience something...particular audience and venue before tailoring each presentation appropriately.  Find out who will be listening...

...phosphorylated residue of interest is mutated to a negatively charged residue, aspartate or glutamate, imparting the...Additionally, these mutations are not reversible, and thus cannot reflect the true state of protein modification...active through phosphomimetic mutation of these residues. Due to the limitations previously mentioned, phosphomimetic...phosphomimetic mutations on the activation loop do not fully activate MEK1. In fact, replacing just one...activity over 70-fold over dual phosphomimetic mutation. This emphasizes the advantages of bona fide pSer-incorporation...recombinant proteins as an alternative to phosphomimetic mutation. Creating phosphoserine-incorporated proteins ... residue that wasn’t originally a serine (e.g. mutating a threonine residue to a phosphoserine) to control...

...microscopy are as follows: Detailed documentation, which facilitates the reproduction of the structure ...are constantly being developed which combine computational processing with biological protocols and revolutionary...creativity and feedback from the community to create state-of-the-art devices for research. There are a number...algorithms for analyzing time-lapse images, quantitatively evaluating antibody staining, or counting cells...Machine-learning denoising of raw data (CARE), segmentation of imaging data (ILASTIK), or the popular plugin-based...trying to overcome this hurdle by taking these limitations into account in the development process. Widely...assemblies open-source. The sharing of digital representations of the 3D models, which in many cases can be...

...and the canonical loxP sequence is ATAACTTCGTATA-GCATACAT-TATACGAAGTTAT. The loxP sequence does not occur...sequence. Among these are loxN (GtATACcT), lox2272 (GgATACtT), and lox511 (GtATACAT). These variant lox sites... The two loxP sites then align in a parallel orientation, allowing the four Cre proteins to form a tetramer...three general outcomes based on the location and orientation of the loxP sites: Inversion: If the loxP sites...are on the same DNA strand and are in opposite orientations, recombination results in an inversion and the... organ, or bypassing embryonic lethality. To facilitate the use of Cre-lox technology, transgenic mice... separate recombination events? One potential limitation of using loxP sites is the inability to tightly...

...offers an opportunity to summarize the limitations of current state of the art technologies. Therefore, .... Now is the time to make bold statements. While some of the statements may be slightly far reaching, ...build upon and address some, if not all, of the limitations of current methodologies. This section sets the... why your funded proposal will address these limitations and lead to new milestones in the field. Research...preliminary data suggests the feasibility of the statement, use it to confirm that your solution is the best... goal and the impact of the project are clearly stated and align with the description of the RFA and funding...italic, or underline font formats helps these statements stand out from the rest of the text. Specific...

...the following paths to maintaining a status in the United States including green cards that are well-tailored...looking to continue their careers in the United States. There has been a growing shift away from employers...with filing a permanent residence case with United States Citizenship and Immigration Services (USCIS). If...with working with US immigration in its current state. As of September 2017, USCIS received 116,224 employment...least a bachelor's degree or its equivalent and state licensure, if required to practice in that field...proposed endeavor will be beneficial to the United States For the purposes of evaluating an NIW, USCIS will...objective criteria including awards, media attention, citations to your work, downloads, commercialization of ...

...diseases. Spontaneous mutations are a major cause of rare diseases. Spontaneous mutations that change protein...biosynthetic pathway and have a wide range of clinical manifestations. PMM2-CDG is the most frequent CDG type characterized...deficient phosphomannomutase (PMM) activity due to mutations in the PMM2 gene. Videira’s group recently reviewed...27393411. Michael McMurray: Rescuing arginine mutations in rare disease with guanidine Michael McMurray...different Arg-mutant enzymes could be rescued by supplementation with guanidine hydrochloride (GdnHCl). Rescue...cells for testing in the lab will be greatly facilitated by CRISPR reagents and other specific gene sequences...suffering from rare diseases arising from Arg mutations. References: Baldwin, Enoch, et al. "Generation...

...preps Mapping antibody escape in SARS-CoV-2 by mutational scanning By Seth Kasowitz As multiple antibody-based...coronavirus 2 (SARS-CoV-2), the virus may acquire mutations to escape these therapeutics. Jesse Bloom’s lab... library encompassing all possible amino acid mutations of the receptor binding domain (RBD) of the viral...et al Cell., 2020. (B) Mapping antibody-escape mutations with the yeast display library. From Starr et ...antibodies. In the library, each plasmid encodes the mutated spike protein and a barcode, and is designed so...The Bloom lab used the library to identify RBD mutations which do not interfere with ACE2 binding, but ...aaRSs. They found the antibodies were able to immunoprecipitate the entire complex in three different cell...

...careful balancing of the inherent advantages and limitations of any technique in the context of the biological...reactions that complicate the effectiveness and interpretation of RNAi. Fortunately, researchers studying ...inverted repeats separated by a loop region that facilitates the formation of a hairpin RNA structure, resulting...codons, and/or the phenomenon of transcriptional adaptation (Smits et al., 2019; Sztal and Stainier, 2020...active development, the first step necessarily dictates the direction and scope of the subsequent two....DNA-based vector or purified RNA) and the initial state of the dsRNA upon activating the RNAi machinery ... of long hairpin RNA (hpRNA). This method is facilitated by DNA constructs that enable insertion of ~400...

...provides a list of putative off-target sites that they can later sequence for mutations. One method a researcher...observed phenotype was caused by an off-target mutation and not an edit in the target gene. The validation... predict off-target effects of sgRNAs using computational methods. They identify possible off-target sites...certain that their samples don’t have off-target mutations if they remain undetected using these techniques...techniques. Frequencies of off-target mutations are essential data points for investigators looking to definitively...the most complete information profile regarding putative off-target sites including both the edit rate ...the whole story Figure 1: Schematic representations of the indicated unbiased genotyping options...

.... Remember to insert your DNA in the correct orientation in the recipient plasmid by viewing the MCS and...Forward Primer will use the sequence 5'-ATGTGGCATATCTCGAAGTAC-3' for the region that binds the ORF and...primer, making our Forward Primer 5'-GAATTCATGTGGCATATCTCGAAGTAC-3'. Many restriction enzymes do not cut...final Forward Primer sequence of 5'-TAAGCAGAATTCATGTGGCATATCTCGAAGTAC-3'. For the Reverse Primer, the design...of the ORF, including the stop codon (5'-TGGCATATCTCGAAGTACTGA-3'), then adding NotI (GCGGCCGC) and then.... This gives us a sequence of 5'-TGGCATATCTCGAAGTACTGAGCGGCCGCTAAGCA-3' (30bp with 18bp of homology to...chose for our reverse primer (5’-TGGCATATCTCGAAGTACTGAGCGGCCGCTAAGCA-3’) into this calculator we get a...