...following four components into 293T cells. Figure 1: The key components of Nanoblades. Image from Mangeot...protein. A single Nanoblade particle can be loaded with 1-4 different gRNAs. Viral envelope proteins To alter... between ~50-70% for these four cell types (Table 1). Additionally, hiPSCs and mouse BM cells still behaved...hepatocytes  Myd88  ~50% Human HSCs  Myd88  ~50% Table 1: Nanoblades editing efficiencies of primary cells....accessible genome editing tool.   References Mangeot, Philippe E., et al. "Genome editing in primary cells and...

... the example shown in Figure 1, large sequences corresponding to exons 1 and 3 of the targeted gene have...knock out exon 2 of a given gene.   Figure 1: A knockout targeting vector designed to insert a ... the Neomycin resistance (NeoR) gene between exon 1 and 3 of the targeted gene. Upon recombination, the...can be used to replace genes in yeast, mice, or Drosophila through homologous recombination. The concept...

... @Addgene congrats for 1 million orders. @RothLabUNC accounts for 10,000 or 1%! — BryanRoth (@zenbrainest... scientific sharing community (check out the infographic below for stats on our plasmid deposits and requests...

...on-target and off-target editing efficiency Figure 1: Effects of (A) double and (B) single base pair gRNA-target...that Cpf1 can tolerate mismatches at gRNA positions 1, 8, 9, and 19-23. Accordingly, the 3’ end of the gRNA...Most gRNAs directed low-frequency Cpf1 cleavage at 1-12 off-target sites; in contrast, SpCas9 may cleave...fits into the genome editing landscape. References 1. Kim D, Kim J, Hur JK, Been KW, Yoon SH, Kim JS. Genome-wide... off-target editing rates, determined using sophisticated genome-wide analysis. At many of its computationally...

...attract fish for them to prey upon (Figure 1). Figure 1: The sea anemone's red color can be used ...synthetic biology. Journal of Biological Engineering, 12(1). https://doi.org/10.1186/s13036-018-0100-0 Murakoshi...expression in living cells. Scientific Reports, 9(1). https://doi.org/10.1038/s41598-019-48604-4 Salih...surface of the chromoprotein Ultramarine with hydrophilic amino acids to reduce non-specific interactions...

...discovered such as Kaede and PS-CFP2 (see Table 1). Table 1: Properties of Selected Irreversible Photoactivatable...) by mutating Threonine 203 to Histidine (T203H) (1). wtGFP normally contains a mixed population of neutral...in fluorescence when illuminated with blue light (1). Similar to PA-GFP, scientists later developed red...fixation conditions to preserve cellular structures (0.5-1% osmium tetroxide). These conditions destroy most ...with extraordinary molecular detail.   References 1.Patterson, George H., and Jennifer Lippincott-Schwartz...is believing." Annu. Rev. Biomed. Eng. 10 (2008): 1-38. PubMed PMID:18647110. Additional Resources on ...reversible photoactivation - and have enabled sophisticated imaging techniques.     Irreversible photoactivation...

...Transposons: The jumping genes. Nature Education 1(1):204. https://www.nature.com/scitable/topicpage/transposons-the-jumping-genes... They transpose by a ‘copy-paste’ mechanism (Fig. 1). They first copy themselves as RNA transcripts and...their host’s genome.   There are two types of Class 1 TEs: those with long terminal repeats (LTRs) and those...reverse transcriptase activities.    Figure 1: Overview of retrotransposon transposition. Retrotransposons...CRISPR-Cas system. This system has four major components: 1) a CRISPR RNA (crRNA), 2) four proteins (TniQ, Cas8...lentivirus). Large cargos, such as the ~11 kB cDNA for dystrophin, the gene mutated in muscular dystrophy, require...

...new combinations of PE protein components (Figure 1), an altered epegRNA delivered with an enhanced pU6cm...amplification of cargo, and heat treatment.  Figure 1: Left: recombined prime editing tools PE2max-NC and...conformational dynamics of the motor protein kinesin-1, the Twelvetrees Lab took advantage of the self-labeling..., Craggs, T.D., & Twelvetrees A.E. (2024) Kinesin-1 is highly flexible and adopts an open conformation...Park, J. C., Zhao, J., Luebbers, A., Green, E., Philibert, C. E., Zhang, H., Layne, M. D., Wu, J. C., & ...

...oxygen. Additionally, its status as a Biosafety Level 1 microbe makes it a perfect candidate for introductory...Transport, biofilms, and much more. Because of it’s BSL-1 status, E. coli can be used as a model to relate these...with superior performance. Nature Communications, 9(1). https://doi.org/10.1038/s41467-018-04899-x Yokoyama..., or frogs, are one of the most popular model amphibians in science with early research providing insight... Jiang, J. P. (2016). Research proceedings on amphibian model organisms. Dong wu xue yan jiu = Zoological...

...central ɑ-helix that can be split into 3 parts: (1) β-strands 1-9 (β1-9) and the one ɑ-helix, (2) β-strand ...apoptosis in HeLa cells, zebrafish embryos, and Drosophila. They treated HeLa cells expressing FlipGFP with...visualize apoptosis of enterocytes in the midgut of Drosophila, an observation that has been difficult until...

... visibility.    Figure 1: Illustration of SARS-CoV-2 from Philippa Steinberg for the IGI.   What...constructs include proteins like nonstructural proteins 1 to 16, spike proteins, envelope small proteins, and...Humana Press, pp 331–338 https://doi.org/10.1007/978-1-59745-181-9_23 Additional resources on the Addgene...

...by mating with Cre-expressing mice. Figure 1: (A) Schematic of the Cre-dependent SPH construct ... alpha (aCaMKII; excitatory neurons) and Synapsin-1 (pan-neuronal) promoters (Figure 1b), and sgRNAs can...ubiquitous expression of sgRNA(s) and mCherry (vector 1) with neuron-specific expression of dCas9-VPR (Figure...Neuron-specific expression is mediated by the synapsin 1 (SYN) promoter, which was superior in its ability ...immediate-early gene Bdnf, which encodes brain-derived neurotrophic factor (BDNF). Bdnf is expressed early in response...

... basic parts of the Cas9 enzyme.   Figure 1: A cartoon depiction of Cas9’s two major lobes, REC...the CRISPR system (Nishimasu et al., 2014). Table 1. Summary of Cas9 domains, the lobe each domain belongs...state (PDB ID 4CMP) with domains colored as in Figure 1. HNH, red; RuvC, orange; CTD, yellow; REC lobe, light... on itself at the bottom of the image. Stem loops 1 and 2 extend horizontally across both lobes. The guide...outside of the protein, spanning both lobes. Stem loop 1, nestled between the REC and CTD domains, is required...Within an interior cavity, Cas9 grips nucleotides 1-10 of the guide RNA, prearranged for base pairing ...343–347. https://doi.org/10.1038/s41586-022-04470-1 Cofsky, J. C., Soczek, K. M., Knott, G. J., Nogales...

...unleashes its cargo – its own genome.   Fig. 1: Viral particle infection into a host cell. Image ...Vector Resources at Addgene! References and Resources 1. Roe T, et al. “Integration of murine leukemia virus...nuclear import of human immunodeficiency virus type 1 preintegration complexes.” Proc. Natl. Acad. Sci. ...approved in the US for multiple diseases, including hemophilia B and spinal muscular atrophy.   Type of Virus...

...true reproducibility. The following are critical: (1) connectivity to other data sources used in research...engagement with the Wisconsin research community were to: (1) prevent vendor lock in by making it easy to get data...other large research institutions. The following graphic depicts the ways in which the two guiding principles...it difficult or impossible to reproduce. As the graphic depicts, RSpace supports linking to all of the ... other tools and resources. As depicted in the graphic, RSpace is integrated with most of the tools noted...

... modify any desired genomic target provided that (1) the sequence is unique compared to the rest of the...will cover three ways to circumvent this limitation: 1) the use of novel S. pyogenes Cas9 variants with varying...Cas9 from Staphylococcus aureus (SaCas9) is about 1 kilobase smaller than SpCas9, so it can be packaged...Biol 19: . https://doi.org/10.1186/s13059-018-1591-1 Cox DBT, Gootenberg JS, Abudayyeh OO, Franklin B, ...Campylobacter jejuni (Cj) NNNNRYAC Streptococcus thermophilus (St) NNAGAAW Treponema denticola (Td) ...

... closely data points fall to each other (see Fig. 1). Alpha values – numerical risk allowance in a ...statistical test assumes your data follows. Fig. 1: Examples of comparable groups with and without equal...values from smallest to largest and ranking them (1 being the smallest value). It calculates the cut off...directly manipulating affect an outcome (age, demographic, diet, etc., in a cancer drug clinical trial,...know type of distribution you are working with, graphing data with a histogram is sometimes sufficient ...

..., including those found at Addgene! Figure 1: Strategy for assembling the barcoded gRNA library...pool-clone them into the pAWp28 storage vector (Figure 1): Forward oligo: 5’- CACCGNNNNNNNNNNNNNNNNNNNGTTTGGGTCTTCGAGAAGACCTATTCXXXXXXXXC... He can be contacted at [email protected].   References 1. Cheng, Allen A., Huiming Ding, and Timothy K. Lu....26190106. PubMed Central PMCID: PMC4559080. 8. Shalem, Ophir, et al. "Genome-scale CRISPR-Cas9 knockout screening...

...Collection Fluorescent tagging strategy Figure 1: Gene editing is the addition of a useful coding sequence...plasmid design strategy. A donor plasmid contains 1 kb homology arms matching the gene of interest, a ...Institute website at alleninstitute.org. References 1. Roberts, Brock, et al. "Systematic gene tagging using...includes systems for tagging mammalian cell lines, Drosophila cells, and C. elegans. In addition to generating...

...want a low affinity calcium biosensor, so that the (1) binding of calcium, (2) allosteric rearrangements... to a red fluorescent state only by simultaneous (1) user-controlled illumination with violet light and...fluorescent indicators. Nature protocols. 2006 Aug;1(3):1057-65. Resources on Addgene.org Addgene’s AAV...GCaMP6 only). The genetic elements required for Drosophila studies have been added for jGCaMP7 and jGCaMP8...

...and drawbacks of four split AAV vector approaches. 1. Overlapping  Overview: The overlapping strategy has...Adeno-Associated Virus Capsids. Human Gene Therapy Methods 27:1–12 . https://doi.org/10.1089/hgtb.2015.140 Frischmeyer...deliver the mini-dystrophin gene to a mouse model of muscular dystrophy. While mini-dystrophin expression levels...levels were lower than expression of wild-type dystrophin in normal muscles, mice treated with the overlapping... trans-splicing vector to deliver a 6 kb mini-dystrophin gene to a mouse model of muscular dystrophy. ...achieved ~80% of muscle cells expressing mini-dystrophin.  3. Hybrid  Overview: The hybrid approach combines...Gene Therapy of mdx Mice With Large Truncated Dystrophins Generated by Recombination Using rAAV6. Molecular...

...reconstitution with a normally functioning T cell repertoire (1). In this example, it was possible to transduce the...significant improvements in vision over a period of 1 to 3 years – the treatment appears to be both safe...research and science communication.     References 1. Gaspar, H. B., S. Howe, and A. J. Thrasher. "Gene...directed at diseases including but not limited to hemophilia B, LPL deficiency, Cystic fibrosis, Muscular ...

...Zhang et al., 2015). In this system a modified TIR-1 protein can degrade degron-tagged proteins in an auxin...splitting cGAL4 in two and binding each half to a gp41-1-N-intein. Split inteins are protein domains that naturally...mutations with unc- 119 + selection." MicroPublication 1 (2018): 2–3. Wang, Han, et al. "cGAL, a temperature-robust...by members of the worm community but there are Drosophila and zebrafish articles published there as well...

...optimized for expression in E. coli: acoustic gene 1 (arg1) and acoustic gene 2 (arg2). The key difference... plasmids are available from Addgene! References 1 Bourdeau, R.W., Lee-Gosselin, A., Lakshmanan, A., ... used arg1 to in vivo image tumour-homing S. typhimurium strain ELH1301. Bourdaeu et al see this as just...

...the other (Sandomenico et al., 2020). Figure 1: Comparison between the IgG antibody and scFv. ...intensity of a tagged protein. SunTag has two components: 1) a protein of interest that’s tagged with 10-24 copies...Application. Clinical and Developmental Immunology 2012:1–15 . https://doi.org/10.1155/2012/980250  Bates A,...without disulfide bonds, made by molecular evolution 1 1Edited by I. A. Wilson. Journal of Molecular Biology...made up of glycine and serine with dispersed hydrophilic residues for increased solubility (Monnier et...

...biosynthesis in Drosophila melanogaster." Molecular and cellular endocrinology 215.1-2 (2004): 1-10. PubMed...stories. Six spooky science stories Zombie ants Ophiocordyceps unilateralis is a fungus that infects ants ... a set of cytochrome P450 genes discovered in Drosophila melanogaster (Gilbert, 2004). Mutations in these...blood out. Once the blood is depleted, the tongue atrophies and falls off. The louse remains becoming the ...

...This gives each guide a score ranging between 0 and 1. The cutting frequency determination (CFD score) is...converted to a weight (which ranges between 0 and 1) so that they can be used in the rank equation above...converted to a weight (which ranges between 0 and 1) (shown in the graph below) so that it can be used...HDR. Optimal mutation in-depth One the most sophisticated features of CrispyCrunch is how it mutates guide...

...biological questions. The two most common uses are: 1) to test the expression level in a specific system...brightness is compared to that of EGFP which is set as 1. Some proteins are very dim (e.g. TagRFP657, which...Photostability can be as short as 100ms (EBFP) or as long as 1 hour (mAmetrine1.2). However, for most FPs it is a...emission is green or red light. For example, T-Sapphire, LSSmOrange, and LSSmKate. Fluorescent Sensors...

...References 1. Peters, Nathan C., et al. "In vivo imaging reveals an essential role for neutrophils in leishmaniasis...do researchers track fluorescently labeled cells? 1. Epifluorescence microscopy This technique uses a ... The video above depicts the infiltration of neutrophils expressing LysM-GFP (green) into a draining lymph...infection with S. aureus (red). You can then see neutrophils swarming around S. aureus and eating the bacteria...two-photon microscopy include: How do T cells and/or Neutrophils travel to the sites of infection or damage (Peters...Good two-photon probes when using a standard Ti:Sapphire Laser include: Blue-green fluorescent proteins...this requires other lasers than the standard Ti:Sapphire laser. It is also important to finely tune the...

...organoid model (Sun, et al. 2021).  Figure 1: Example schematic from the Organoid EasyTag system...without altering that of the internal control (Fig 1). Because this method relies on the fluorescence ratio...and can be used for genome editing in moderate thermophiles.  These evolved SpCas9 variants have improved...

...more tools for plasmid mapping and design (Figure 1). Benchling: While you might think of Benchling as...Davis from the University of Utah.   Figure 1: A plasmid map created in SnapGene of Blue Flame plasmid...patterns, post-translational modifications and polymorphisms Codon optimization tool: To access this tool...

.... In literary terms, conflict can be defined as (1) the struggle between two opposing forces or (2) the...journey even when they know the ending.    Fig. 1: We all know that the superheroes win in the end. ...other affected people, man/animal vs nature, anthropomorphized animals, or even seemingly unrelated stories...

... that dimerizes upon exposure to blue light (Fig. 1). Another blue light-inducible dimerization domain...expression needed for most applications.   Figure 1: With blue light, the VVD domains of the BLADE dimerize...Mustafa Khammash's lab at ETH Zürich. “Instead of a graphical abstract, it’s a musical abstract,” says Di Ventura...

...tethered to bring them into close proximity (Figure 1). Formation of the complete FP results in a fluorescent...detected by fluorescence microscopy. Figure 1: How BiFC works. A split FP (e.g., yellow fluorescent... protein interactions in living cells. Nat Protoc 1:1278–1286. https://doi.org/10.1038/nprot.2006.201 ...analyzing protein–protein interactions in live Drosophila. eLife 7. https://doi.org/10.7554/elife.38853...

...experiments or even negatively impact cells (see Figure 1). When behaving properly, FPs enable researchers ... disulfide bonds in the secretory pathway (Figure 1) [4,6–8]. Additionally, FPs that encode N-glycosylation... sarcoma-associated herpesvirus.       References 1. Snapp, Erik. "Design and use of fluorescent fusion...genes: deletion mapping functional domains of Drosophila hsp 70." The EMBO journal 3.13 (1984): 3087. ...

...another great year of sharing!   References 1. Shalem, Ophir, et al. "Genome-scale CRISPR-Cas9 knockout...Central PMCID: PMC4089965. 2. Sanjana, Neville E., Ophir Shalem, and Feng Zhang. "Improved vectors and genome-wide...

...mediating the entry of SARS-CoV and SARS-CoV-2 viruses. ( 1 ) TMPRSS2 - a serine protease that primes the SARS-CoV...transcriptase expression plasmids from the Drew Endy and Philippa Marrack Labs. Point-of-care testing for COVID-...

...sequences were initially discovered in E. coli in 1987 (1), the concept that these clustered repeat sequences...neutralize CRISPR/Cas. Earlier this year, expression of 1 of 5 anti-CRISPR genes were found to inactivate the...light until 2007. Initial experiments exposed S. thermophilus to predatory phages to test if the exogenous...

...has error rates that range from roughly 1 per 500bp to roughly 1 per 10 million bp depending on the polymerase...analyzing the gene’s function. The process is shown graphically in the following figure, in which we add EcoRI...

...vectors and include a Kan, SpHIS5, or CaURA3 selection marker. Lee et al.(1) assessed many of these fluorescent... Shah NG, McKenzie A 3rd, Steever AB, Wach A, Philippsen P, Pringle JR. Yeast. 1998 Jul;14(10):943-51....3rd, Demarini DJ, Shah NG, Wach A, Brachat A, Philippsen P, Pringle JR. Yeast. 1998 Jul;14(10):953-61....