... selecting, testing, and optimizing three key variables of an AAV vector: serotype, promoter, and titer...steps in testing are effected by multiple AAV variables.    Serotypes Serotype refers to the different...

...work, there are other options. Commonly used Cpf1 variants, with a  5’ TTTV PAM, improve targeting in AT-...respectively. Researchers also engineered SpCas9 variants with alternative PAMs like NGAG and NGCG and SaCas9... in the pool. According to this logic, ω-dCas9 variants recognizing multiple PAMs should have a fitness...promoting survival of only the broadest-targeting variants. For the final round of evolution, they used a... a no-G PAM library to enrich for Cas9 variants with activity on non-NGG PAMs, generating xCas9 3.0-3.13...confirming the flexibility of xCas9 3.7. Base editor variants with xCas9 are similarly flexible. Like the xCas9...References Hu, Johnny H., et al. "Evolved Cas9 variants with broad PAM compatibility and high DNA specificity...

...no one option is perfect, with all this variance in your variant options, you’re likely to find an application...It is a well known fact if a multicopy variant of a gene exists, there must be assays that researchers...quantify said copies. They do so using copy number variation (CNV) assays. There are several varieties of these...small as reasonably possible and set using the variance seen within multiple runs of the standard curve...identify trends in cases where the copy number variation is two or less (Muhamad, 2011). Ambiguous Taqman...Usher CL, McCarroll SA. Analyzing Copy Number Variation with Droplet Digital PCR. Methods Mol Biol. 2018...Chung WK. Quantitative analysis of copy number variants based on real-time LightCycler PCR. Curr Protoc...

...single polypeptides that contain the variable light chain (VL) and variable heavy chain (VH) of an antibody...technology. But what about single chain fragment variables (scFvs)? scFvs weren’t developed for another decade... 2013). The linker keeps the C-terminus of one variable domain and the N-terminus of the other domain ...oligomerization of the scFv. While you might assume that the variable domains of an scFv would mirror that of an antibody...screen for new antibodies. To do this, antibody variable domain sequences are expressed as scFvs and screened...for strong binders. Then the sequences for the variable domains of the selected scFvs are inserted into...2013) In Vivo Applications of Single Chain Fv (Variable Domain) (scFv) Fragments. Antibodies 2:193–208...

...are delivered by feeding them to planarians. Depending on which planarian species one is working with, the...accessible or too challenging for amateur scientists. Planarians, wormy creatures commonly found in freshwater...provide a good potential solution to this problem. Planarians are easy to buy, cultivate, and have interesting...advanced developmental biology experiments in planarians with their recent plasmid deposit. These plasmids...in four inexpensive and commercially available planarians: Girardia sp., Dugesia dorotocephala, Phagocata...into plasmids to investigate for each of these planarian species. These plasmids all have a PR-T4 vector...technique. The expression patterns of 11 different planarian genes (all of which are available through the ...

...-type. However, variants such as the P.1 (Gamma variant) and the B.1.351 (Beta variant) were much less...vaccine could neutralize many variants, including the B.1.1.7 (Alpha variant), B1.1.298, and B.1.429 (Epsilon...on the variant. The lowest levels of cross-neutralization occurs in the P.1 and B.1.351 variants tested...circulating variants. This allowed us to create pseudoviruses that represent lineages and variants that are...luminescence.     B.1.351 variant neutralization escape The lab saw that the B.1.351 variant was very resistant... distinct from SARS-CoV-2 and its variants. Yet, the B.1.351 variant, which has only 9 different mutations...we’ve seen the emergence of several SARS-CoV-2 variants coincide with increased vaccination efforts. So...

...effects of sequence variants on abundance simultaneously called VAMP-seq, for Variant Abundance by Massively...the abundance of a protein variant library is monitored by fusing the variants to eGFP followed by an internal...Quantifying the abundance of thousands of protein variants simultaneously  With increasing sequencing power...individual’s genome. But when they find genetic variants, what does that mean? Previous research shows ...approximately 75% of all protein-coding pathogenic human variants are decreased in protein abundance, possibly due... due to misfolding. But measuring variants one at a time is time consuming and unscalable.  Melissa Chiasson...Washington sought to address the effects of genetic variants in a high throughput manner. She helped develop...

...devised (8). References 1. Casadaban M.J., Martinez-Arias A., Shapira S.K., Chou J. Beta-galactosidase gene...lacZ reporter gene (3, 4), there are now many variations, including many that use auxotrophic rescue genes...

..., F., Lach, R. S., Baxter, N. J., Kosik, K. S., Arias, C., Acosta-Alvear, D., & Wilson, M. Z. (2020). ...also a good way to diagnose single nucleotide variations. Also, FnCas9 cleaves in a range of temperatures...

...J. D., Pelkey, K. A., Chittajallu, R., Guo, B., Arias-Garcia, M. A., Allaway, K., Sakopoulos, S., Schneider...unique tropisms are now available. These include variants with broad CNS or PNS transduction, cell type ...Tabebordbar et al., 2021) method, selecting for variants that were effective in C57BL/6J mice, BALB/cJ ... by transferring the 7-mer peptide region from variable region VIII of AAV9-X1.1 to that of AAV1. AAV1...BALB/cJ and C57BL/6 mice and rats. In mice, no variation in transduction efficiency was seen between different...lower than those seen with AAV9, they were highly variable and were not lower than those of AAV-PHP.eB (Goertsen...resources on the Addgene blog Viral Vectors 101: AAV Variables That Matter Viral Vectors 101: An Introduction...

...Independent variables – factors which are not affected by other variables (cause). Dependent variables – factors...assumptions: Distribution – Normal Variables – Independent Variance – Approximately equal Data type - ...two (two-way ANOVA) independent variables on a single dependent variable. A two-way ANOVA could test the... the effect of two dependent variables on up to two independent variables. For example, a one-way MANOVA... assesses multiple independent variables on a single dependent variable. I know what you’re thinking –...and MANCOVA All variants of ANOVAs discussed so far assume that the independent variables are independent...have discrete effects on the dependent variable, and those variables are the only things that matter. However...

...individual variants found in tumors are exceedingly rare. As a result, how most of these “variants of unknown...cancer clinical sequencing report includes rare variants that haven’t been characterized? Some have developed...approach this challenge, using attributes of cancer variants such as evolutionary conservation, or predicted...hope researchers can use to characterize these variants in a high-throughput manner and therefore push...precision medicine a reality. Challenges for cancer variant characterization For many years, we’ve hoped that...laboratory-based functional experiments that classify which variants are likely gain-of-function, loss-of-function ...these experiments could find correlations between variants in a gene of poorly classified function and a ...

...four novel SpCas9 variants with altered PAM binding specificity: SpCas9 variant Mutations (relative...SpCas9) PAM sequence D1135E variant D1135E NGG VQR variant D1135V, R1335Q and T1337R NGAN...NGAN or NGNG EQR variant D1135E, R1335Q and T1337R NGAG VRER variant D1135V, G1218R, R1335E...the VQR and VRER variants is similar to wild-type SpCas9, indicating that the variants are likely just ...reported the development of near-PAMless Cas9 variants. These variants, named SpG and SpRY, can target NGN PAMs...limitation: 1) the use of novel S. pyogenes Cas9 variants with varying PAM sequences, 2) the use of Cas9...R1335E and T1337R NGCG The D1135E variant is far more selective for the canonical S. pyogenes PAM...

...try developing a variant with an enhanced response. Evolution of a new variant Maria and the team set ...suggest she deprioritize the project, Maria’s screens of AsLOV2 variants showed some significant gains! After...several other labs. Andrew and Maria expect that further-improved variants of MagLOV will be possible with...applications that don’t even exist yet? Andrew York, Maria Ingaramo, and their team at Calico Life Sciences...right through our tissues unimpeded. Andrew and Maria dreamed about the possibilities of a magnetoresponsive...appears to be weak — if they exist at all. Andrew and Maria were encouraged by previous reports that described...developing useful tools. A lucky start As Andrew and Maria explained, “to our surprise and delight,” the very...

...Campbell’s lab added two new mCherry variants to the repository. These variants can be used as tools to learn... Podcast Segment The impact of KLF4 N-terminal variants on iPSC generation Deriving iPSCs (induced pluripotent...polycistronic cassette. However, it was unknown whether variations in gene expression from these polycistronic cassettes...transposons, the Woltjen Lab compared different variants of the polycistronic cassettes and discovered ...which they traced to a 9 amino acid N-terminal variation in the Klf4 isoform (KLF4s or KLF4L). Polycistronic...discovered that monocistronic expression of either variation of Klf4 did not lead to the observed reprogramming... developed a synthetic red fluorescent protein variant they call mScarlet. Bindels et al used a combination...

...green (v857) variants differing in kinetics, affinity, and ∆F/F0. Introduce the two variants in three different...for sfGFP to create SF-iGluSnFR. This improved variant has higher expression levels in bacteria and produces...models. Furthermore, they created three affinity variants – A184S (increased affinity for glutamate), S72A...Kaspar Podgorski’s lab have recently made iGluSnFR3 variants that are more sensitive and better localized for...predecessors. The detailed characterization of the new variants is available here. Upgrades to iGluSnFR3 To improve...Improved photophysical properties The improved variants of iGluSnFR3 have higher expression levels and...   Increased versatility through two different variants The yellow (v82) and lime green (v857) versions...

...' TTTV AsCpf1 RR variant 5' TYCV LbCpf1 RR variant 5' TYCV AsCpf1 RVR variant 5' TATV Campylobacter... PAM sequence. Cas9 Species/Variants and PAM Sequences Species/Variant of Cas9 PAM Sequence ...NGG SpCas9 D1135E variant 3' NGG (reduced NAG binding) SpCas9 VRER variant 3' NGCG SpCas9...SpCas9 EQR variant 3' NGAG SpCas9 VQR variant  3' NGAN or NGNG  xCas9 3' NG, GAA, or GAT SpCas9...general overview of CRISPR components, new Cas9 variants are being discovered all the time and the requirements...different systems can vary (for example, xCas9 is a variant with increased PAM flexibiliy and eSpCas9/SpCas9...protein required is the Cas9 endonuclease or a variant thereof. This individual protein has all the components...

...controls are invariably inconclusive and unreliable. Proper controls provide the constant variables that enable...independent variable. Referring back to the experiment associated with Figure 1, the independent variable is the...interpretation of the effect of the independent variable you are testing. Importantly, they demonstrate...is specifically associated with the independent variable you are testing and not some other unintended ...to minimize any effects of the non-independent variables.  To illustrate the importance of control plasmids...to minimize the effects of the non-independent variables within an experiment, both the design and selection...experiment and the interrogation of the independent variable. Replicate controls: technical and biological ...

...library of AAV9 variants (Chuapoco et al., 2023). They found the best-performing variant, CAP-Mac, was ...genome editing for primary cells Monomeric StayGold variants AAV.CAP-Mac for systemic gene transfer in primate... Breaking up is hard to do: Monomeric StayGold variants By Mike Lacy In 2022, Atsushi Miyawaki and colleagues...stability. Here’s a quick overview of these new variants:   Figure 2: Inner mitochondrial membranes...QC2-6 FIQ or mStayGold (Ando et al., 2023). This variant has similar photostability and brightness compared...Ivorra-Molla et al., 2023). They also named their variant mStayGold. It is worth mentioning that Kiryl Piatkevich...colleagues generated yet another monomeric StayGold variant (Piatkevich et al., 2023). Check out their preprint...

...downstream of the variable region gene in this order: μ, δ, γ, ε, and α. An antibody variable gene is initially...heavy chain, light chain, constant region, and variable region.   Why are antibody isotypes important... heavy chain constant domain it pairs with its variable chain domain. B cells start by co-expressing IgM...antibodies, but later in an immune response, the variable region of an antibody is paired with any of the...intervening DNA sequence is deleted, which places the variable region next to a different constant domain gene...heavy-chain constant domain genes, placing the variable domain genes next to a different constant domain...the B cell expressing an antibody with the same variable domain but a different heavy-chain constant domain...