...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...rate as SpCas9 at an NGG PAM. When they tested a variety of NG, GAT, GAA, and CAA PAMs, they saw that xCas9...confirming the flexibility of xCas9 3.7. Base editor variants with xCas9 are similarly flexible. Like the xCas9...

... 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...numbers for context. That’s because the number can vary depending on your route of administration, what ...., Solovyeva, V. V., & Rizvanov, A. A. (2023). Various AAV Serotypes and Their Applications in Gene Therapy...

... do so using copy number variation (CNV) assays. There are several varieties of these, but in this blog...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...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...

...-type. However, variants such as the P.1 (Gamma variant) and the B.1.351 (Beta variant) were much less... the variants? Alejandro Balazs, a Group Leader at the Ragon Institute of MGH, MIT, and Harvard, and ...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...

...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...

... (capsid) N and C 79633 (N) and 79641 (C) NS1 (var: W98G) N and C 79635 NS3 C 79637 NS2B C 79636... activity have remained challenging due to the variable availability of enhancer sequences in different...the entire genome for genes that regulate a wide variety of phenotypes, including but not limited to cell...CIDAR MoClo) Parts Kit. This Kit consists of a variety of plasmids containing many different promoters...RBS’, coding sequences (CDS), and terminators of varying strengths. These can be easily combined with your...creation of many E. coli constructs with greatly varied levels of expression. The 93 plasmids in the CIDAR...gene was chosen to represent the primary splice variant in 38 types of human cancer. Many of the transcripts...

...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...of the Cancer Program at the Broad Institute of Harvard and MIT. The notion of cancer precision medicine...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 ...

...limitation: 1) the use of novel S. pyogenes Cas9 variants with varying PAM sequences, 2) the use of Cas9 homologs...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...R1335E and T1337R NGCG The D1135E variant is far more selective for the canonical S. pyogenes PAM...

...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...Dlx5/6) enhancer elements and combined it with a variety of reporters (i.e. eGFP) and effectors (i.e. DREADDs...

...' TTTV AsCpf1 RR variant 5' TYCV LbCpf1 RR variant 5' TYCV AsCpf1 RVR variant 5' TATV Campylobacter... of these different systems can vary (for example, xCas9 is a variant with increased PAM flexibiliy and... 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... will be able to describe the functions of the various CRISPR/Cas9 components. Please note that while ...general overview of CRISPR components, new Cas9 variants are being discovered all the time and the requirements...

...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...every day. Although the designs and outcomes may vary, one thing should be present in every experiment-based...interpretation of the effect of the independent variable you are testing. Importantly, they demonstrate...your experiment. Read on to learn more about the various controls that can be used for plasmid-based experiments...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...

...green (v857) variants differing in kinetics, affinity, and ∆F/F0. Introduce the two variants in three different...and other brain cells in vivo. Glutamate plays a variety of roles in synaptic communication and can trigger...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...

...protocol may vary depending on your specific experiment, but a broad overview of the various ways in which...your experimental target, SpCas9 and its enhanced variants, eSpCas9 and HypaCas9, are great choices. Check...our SpCas9 containing plasmids, optimized for a variety of species.   Specificity concerns? If off-target...regions of the genome and consider using a Cas9 variant with higher specificity (eSpCas9 and HypaCas9) ...added bonus of low off-target effects. The SpG variant, discovered by the Kleinstiver group has a PAM ...Further, this group also characterized a second Cas9 variant, SpRY, which cuts at NRN PAMs with high efficiency...a small window, this system can be used with a variety of impaired Cas9 proteins (such as SpRY) with altered...

...of interest in a variety of model systems, thereby opening your research to a variety of experimental ...sequencing (pInitial) and protein production (a variety of pExpression vectors) plasmids. The type IIS .... Expression vectors within this kit contain a variety of promoters to facilitate gene expression in bacteria...oocytes. The expression vectors additionally contain various combinations of protein tags (His, Avi, and GFP...plasmids in this kit have been used to purify a variety of peptides including antibiotic efflux pumps and...created this kit. The plasmids within it contain a variety of different promoters followed by leader sequences... at the 3’ end of the leader sequence. Using a variety of GFP and RFP fusions as their test genes, the...

...’s lab in Novartis Oncology.  Bhang did her PhD work at Johns Hopkins and came to Novartis for her post-doc...research. When she came to Novartis, she was happily surprised that “Novartis was actually really research...This article is based on an interview with Novartis researcher, Carrie Bhang. The ClonTracer Library,...investigator in the In Vivo Pharmacology group at Novartis Oncology, is an exciting new tool that allows ... and says that she decided to do a post-doc at Novartis because she wanted first-hand experience in industry...co-workers started using the tool to look at a variety of questions surrounding cancer development, metastasis...investigator in the In Vivo Pharmacology group at Novartis Oncology, Bhang has been working on using the ...

...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...

...immune system and a tool that can be used for a variety of research applications. Within the immune system...regions are completed with the Ig light chains. The variable region of the heavy and light chain determine ...Fab, heavy chain, light chain, constant region, variable region   The constant region of the heavy chain...antibody depends partially on its isotype. The variable region of an antibody does not recognize the entirety...antibodies are extracted. These antibodies recognize a variety of epitopes on the target protein, so they are ...antibodies against different protein epitopes and so variability between lots is a big concern. You can find more...clonal population of cells that produce a single variant of antibody. Monoclonal antibodies are less likely...

...naturally-occurring variations in the TALE repeat, referred to as “non-RVD variations”, were found to improve...successfully create GFP fusion proteins with a variety of genes across the genome. Although this tool ...characterization, has enabled scientists to engineer a wide variety of GFPs with diversity in fluorescence brightness... engineering, Ed Boyden’s lab has identified a variety of new channelrhodopsins for use as optogenetics...which is 45 nm more red-shifted compared to other variants. The group further optimized Chrimson through ...off-kinetics (15.8 ± 0.4ms from 21.4 ± 1.1 ms); this new variant was named ChrimsonR. Plasmids containing Chronos...lentiviral and AAV expression vectors for certain variants. Klapoetke et al., Nat Methods 2014 Mar 11;(...