...Kim et al., 2017 SaBE3, BE3 PAM variants, BE3 editing window variants   Greatly expands the number of ...increased editing frequency to above 30% for a variety of targets in human cell lines, with an average...upstream of the PAM.  David Liu’s lab generated BE3 variants with other deaminases: AID, CDA1, and APOBEC3G... Liu lab also used natural and engineered Cas9 variants to develop five new base editors with distinct...HF-BE3, a base editor containing high fidelity Cas9 variant HF-Cas9 (Rees et al., 2017). HF-BE3 showed 37-fold...Gaudelli evolved the base editor into 40 new ABE8 variants (Gaudelli et al., 2020). Compared to ABE7.10, ...-fold higher editing efficiency at non-NGG PAM variants compared to ABE7.10. ABE8s have an improved base...

...observations of oxytocin dynamics in live mice under a variety of behaviors, opening up new research directions...other MTRIA sensors based on GPCRs recognizing various neurotransmitters, neuropeptides and other ligands...circularly-permuted GFP into an intracellular loop of various G-protein-coupled receptors. Image adapted from...for synaptic glutamate response. They generated variants optimized for different applications (v82 has ...faster kinetics but lower affinity) and with a variety of tags for localization (PDGFR, GPI, and SGZ).... and powerful high-throughput screen to select variants based on performance metrics specifically for ...using potassium-binding protein homologues from various bacteria led to sensors of intermediate affinities...

...Crystal Shih is an Investigator III at Novartis, a multinational pharmaceutical company based in Switzerland.... Crystal now works on protein therapeutics at Novartis - listen to learn more about Crystal’s exciting...Crystal's educational background and position at Novartis. Chapter 2: What's a lab group in industry? The...empower people Chapter 3: How Crystal advanced at Novartis, and how to prepare yourself for success by having... The types of science that Crystal works on at Novartis, how she's learned to prioritize projects, and...listings. Mobility between different positions at Novartis. Chapter 7: How Crystal developed her science ...communication skills and how they've helped her at Novartis. The importance of mentorship in developing career...

...signal. However, they have high cross-reactivity, vary significantly from lot-to-lot, and have to be re-validated... plasmids and have very little batch-to-batch variation. Confirmation of sequence can be done from relatively...sizes.  Table 2: Recommended gel percentages for various protein sizes Protein Size Target Proteins...capacity and high protein retention. It comes in a variety of pore sizes and protein capacity, allowing you...lot of work around a flame. Membranes come in a variety of pore sizes. If your protein of interest is small...dry/electroblot transfer systems available from various manufacturers. These proprietary systems are fast...are worth considering if your proteins of interest vary wildly in size. However, these systems are expensive...

...to try developing a variant with an enhanced response. Evolution of a new variant Maria and the team set... clear that this was even possible. There is a variety of research on whether and how organisms respond...mutagenesis and screening, the EGFP and mScarlet variants were showing no obvious signs of improved response...deprioritize the project, Maria’s screens of AsLOV2 variants showed some significant gains! After a few more...the magnetic field (Figure 2); they named this variant MagLOV. Andrew was thrilled to see that MagLOV’...: Fluorescence intensity and ΔF/F of MagLOV, a variant of AsLOV2 with mutations C450P, L496V, Q513K, G528K... Andrew and Maria expect that further-improved variants of MagLOV will be possible with other engineering...

...levels.   Overview of Variations Between Brainbow Systems   Brainbow variants and applications beyond... enables Brainbow to be used in a variety of species. More variants have been created by other labs, ...2007, Joshua Sanes and Jeff Lichtman, both of Harvard’s Department of Molecular and Cellular Biology, ...expression; these deletions are defined using 2 loxP variants (only identical loxP sites can mediate recombination...either deletion removes one of the other loxP site variants, preventing further recombination. Brainbow-1.1...with recombination events specified by three loxP variants, but the principles remain the same. The first...expect ten colors (see table to the right); in various reports, Sanes and Lichtman have observed from ...

...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...

...’ll dive into the opposite: AAV serotypes with varying tissue tropism, allowing them to target particular...shell packages the vector’s genetic material, and variations between serotypes can change how the viral particle...be present on different cell types and sometimes vary between target species or even strains. Pro tip!...serotypes in this post. However, tissue tropism varies both between species and even between strains of... mice in vivo. Though tropism depends on many variables, we suggest these serotypes as a good place to...applications. Cross-reactivity of AAV serotypes varies by species and route of administration (Wu, Asokan...., Solovyeva, V. V., & Rizvanov, A. A. (2023). Various AAV Serotypes and Their Applications in Gene Therapy...

...very soon. Not only is CRISPR having effects on various biological disciplines, the base technology itself...itself is constantly improving. Cas9 variants have been modified for genome editing, activating gene expression...specificity of the Cas9 nuclease with engineered variants: eSpCas9, SpCas9-HF1, & HypaCas9. The off-target...off-target effects” have included using a Cas9 nickase variant, lowering Cas9 expression, and truncating gRNA ...Zhang lab at the Broad Institute, the Joung lab at Harvard Medical School, and the Doudna Lab at Berkeley ...of the HNH/RuvC groove, Slaymaker et al. made a variety of alanine substitutions throughout the groove ...off-target cuts when compared to WT SpCas9 across a variety of genomic sites. Knowledge of Cas9’s dynamic structure...

...is supplied on its own expression plasmid or in various combinations, while the cognate att sites are supplied... BRET ratio), which allows for compensation of varying fusion protein expression levels. Zhang et al...ensures that all screened plasmids contain a gene variant* and is amenable to both high and low throughput...protein with cell specific expression containing a variety of N- and C- terminal tags. The Santalucía Lab ...specifically express your new chimeric protein in a variety of model systems. The MultiSite Gateway Kit contains...constructed with the MuLE toolbox have been used for a variety of experimental purposes including (1) single or...been used to study transport characteristics of various Golgi and plasma membrane reporters as well as ...

...exciting year for genome engineering research. A variety of new tools came out including the single base...base editors, Casilio, CombiGEM, and a variety of pooled libraries. Not all of these technologies were without... use to express both SpCas9 and your gRNA in a variety of mammalian cell types. Purpose  SpCas9 This plasmid...CAG promoter that the Zhang lab has tested in a variety of mammalian cell lines. Cas9, of course, is the...particular backbone can be used to transfect a variety of mammalian cells and can be modified for viral...express both SpCas9 and your gRNA of interest in a variety of mammalian cell lines. Alternative forms of this...GFP-tagged and un-tagged versions of SpCas9 and further variants replace SpCas9 with SpCas9n (nickase), which nicks...

...MIT, Alvin Hsu Harvard University, and David R. Liu from the Broad Institute and Harvard University. Over...MIT, Alvin Hsu Harvard University, and David R. Liu from the Broad Institute and Harvard University. Max...sophisticated analyses, we first visualized our data in various ways. Smoothing our plasmid order data with a 7...parameter. We approximated the data likelihood with multivariate Gauss-Hermite quadrature, since the analytical...intractable integral of the product of a Poisson and multivariate log-normal likelihoods. Effects of weekends...applications.       Alvin Hsu is a graduate student at Harvard University. He is interested in using selection...Professor of Chemistry and Chemical Biology at Harvard University; and Howard Hughes Medical Institute...

... simple hierarchy: class to type to subtype to variant. Despite the seemingly complicated steps to arrive...on main function and composition; subtypes and variants are typically based on species of origin. There...currently two classes, six types, 33 subtypes, and 17 variants. Figure 1: Overview of the CRISPR classification...hierarchy flows from Class to type to subtype to variant (shown in italics). Created with BioRender.com....machinery from other CRISPR systems to carry out various functions. Class 2 Class 2 CRISPR systems rely ...others have become more notable in recent years. A variant of V-U, paradoxically called V-K, is a CRISPR-associated...that can insert large fragments of DNA. The V-F variants, which are among the smallest Cas enzymes (400...

...measure the frequency of each variant during the selection process. Each variant is assigned a functional ...of a plasmid library that expresses all desired variants of a protein. Applying a selective pressure winnows...winnows the pool down to plasmids expressing variants with optimal function. High-throughput DNA sequencing...mutational scanning Hundreds of thousands of protein variants can be tested in one experiment. There are many...experiments, libraries of DNA fragments that contain variable bases (NNN) at any given codon site of interest...opposite wild-type DNA strand is nicked by the BbvCI variant not used in step 1. Then this strand is degraded... and cost. You need at least 100-200 reads per variant, so the more codons you test in one library, the...

... released a new variant of jGCaMP7 (keep reading) and will be releasing more variants in the next few ...few months (see “coming soon”). The 7b variant (pGP-AAV-syn-FLEX-jGCaMP7b-WPRE) exhibits the brightest resting...processes like dendrites and axons. Find the 7b variant of jGCaMP7 in AAV1 at Addgene! See all calcium ...membrane localization and brightness. The F102G variant (pAAV.hSyn-FLEX.iGABASnFR.F102G) is noticeably ...responses to GABA. Find the iGABASnFR and its F102G variant in AAV1 at Addgene! Archon1, a voltage sensor AAV...neurons. 105321  AAV1  pGP-AAV-syn-jGCaMP7c variant 1513-WPRE 7c exhibits high contrast between peak...reduced. 105322  AAV1  pGP-AAV-syn-FLEX-jGCaMP7c variant 1513-WPRE7c exhibits high contrast between peak...

...pathogenic genetic variants have been identified in humans and cataloged in the ClinVar database. Previously... potential to correct only a minority of those variants in most cell types. But prime editing, a CRISPR...innovations by the Liu lab and others have led to a variety of prime editing tools. We’ll summarize the major...optimized for each application, and the efficacy varies widely — it still almost always results in higher...research, the Liu lab created and evaluated 19 PE1 variants with RT mutations known to increase activity, ...Even in PE3 and PE3b, prime editing efficiency can vary widely — so its inventors kept optimizing it. Using...PE2max, PE3max, etc. PE6: Specialized prime editing variants Four years after their initial publication, the...

...-AAV-syn-jGCaMP7c variant 1513-WPRE (105321-AAV1) pGP-AAV-syn-FLEX-jGCaMP7c variant 1513-WPRE (105322-...ul) samples of EGFP-expressing AAV packaged in various serotypes. This collection is now complete! pAAV-CAG-GFP...vectors to more serotypes. We currently package four variants of these biosensors, which are optimized for specific...relative to baseline fluorescence, as the GCaMP7f variant, and is approximately 5-fold brighter than GCaMP6s...GCaMP6s. jGCaMP7f is the fastest responding variant. jGCaMP7b exhibits the brightest resting fluorescence...fluorescence from inactive neurons is reduced. This variant has an approximately 12-fold increase in brightness.... We recently released several recombinases in various serotypes, and will continue to grow this collection...

...the most recent advances and how some of these variants may be appropriate for your specific research ...But Cas9s found in other species can recognize a variety of different PAM sequences. Researchers have also...isolated from different organisms and engineered Cas9 variants that were able to target different PAM sites (... be targeted (Hu et al., 2018).  These altered variants can allow for targeting of your genomic loci of...off-site activity of Sniper-Cas9 to different Cas9 variants using gRNAs of different lengths (Lee et al., ...continue to characterize CRISPR nucleases from a variety of sources, it is likely that the toolkit available...genome, there are more and more Cas enzymes and variants to choose from. While no enzyme is perfect for...

...different plasmids. Harvard’s DRSC/TRiP Functional Genomics Resource Center has various plasmids for overexpression...blogger Jon Chow, an immunology PhD student at Harvard University. In my previous two posts, I’ve described...Random insertion has the advantage that you can get varying expression levels of your transgene in different...comparisons between lines expressing different variants of YFG because the transgenes are found in the...that are anywhere from light yellow to red. The variation in color depends on how transcriptionally active...interest, such as the intestines, can be dissected at various times throughout the lifespan of the fly to determine...Chow. Jon Chow is finishing his immunology PhD at Harvard University. He has had nearly a decade of experience...

...Yes Agarose gel Table 1: Features of various qPCR chemistries. Adapted from Wong, 2018   ...should be 60-65°C For qPCR, the important primer variable is the annealing temperature, not the melting ...reference genes have shown to have as much as sixfold variation in expression levels reported (Biassoni, 2014)...negative error on the other will introduce unwanted variation into your data. Try different types of pipettes... within each set of triplicates to understand variation due to technical error.  Plate readouts can be...type or tubes, sealing method, and other small variations in consumables. Track and record these details...Reference Dyes Whatever your pipetting method, some variation will occur. A passive reference dye, such as ROX...