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  1. Tips for a 1st Time CRISPR User (by a 1st Time CRISPR User)

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    Blog Post
    ...of 0.3, as recommended by the original publication. Pro-Tip: 1. Use multiple gRNAs to target different..., as indicated by the presence of a 428 bp digestion product (lane 2). The undigested (-, lane 1) DNA ...The entire process from start to finish took about 1 month. Cas9 delivery Overview: Before any editing...lentiviral vector, lentiCas9-Blast (52962-LV, Figure 1). Some cells were then used to generate a monoclonal...rest of the cells were frozen down. Figure 1: Cas9 expression in A549 cells transduced with lentiCas9...Western blotting is a loading control. Pro-Tips: 1. It’s all about the MOI. In retrospect I used a way... pool had wildly variable Cas9 expression (Figure 1). This is a problem because Cas9 expression affects...

  2. Viral Vectors 101: AAV Variables That Matter

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    Blog Post
    ...promoters. Gene Therapy, 28(1–2), 56–74. https://doi.org/10.1038/s41434-020-0169-1 Oh, J. Y., Han, J. H., ...AAV Serotypes and Their Applications in Gene Therapy: An Overview. Cells, 12(5), 1–41. https://doi.org/10.3390...constitutive promoters located in adeno-associated viral vectors. PLoS ONE, 9(8), 1–10. https://doi.org/...the selection process. Factors to think about are (1) your experimental conditions and measurements; (2...serotype, promoter, and titer.     Figure 1: Flow chart depicting a) steps for selecting serotypes...injection site than AAV2 (Watakabe et al., 2015) (Figure 1). You’ll need to consider if such properties could...

  3. CRISPR Between the Genes: How to Experiment with Enhancers and Epigenomics

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    Blog Post
    ...chromosome, start, and stop. For instance (chr3, 1, 10) indicates a stretch of DNA at the very beginning of .... Table 1: An emerging family of "GPS for the genome" techniques Technique What It Locates ChIP-seq...clinical applications of CRISPR in the next section. In order to denote the NANOG enhancers from step 1 with...genetic elements like protein, RNA, and DNA (Table 1). As with GPS in the real world, coordinates alone...enhancers with the help of genome math. Figure 1: Genome math illustrated. Intersect (left) and set...histones with “GPS for proteins,” ChIP-seq from Table 1. Because of DNA’s 3D geometry and the chemical properties...experiment to identify the critical NANOG binding sites: 1. Find Enhancers that have NANOG binding sites 2. Design...

  4. Troubleshooting and Optimizing a Western Blot

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    Blog Post
    ...the antibody’s dynamic range. Specifically, the 1:2, 1:1, and No dilution samples all have approximately... doing the purification and cross-adsorption in lab, consider a dilution range from 1:100–1:1,000. If ...recommended dilution and/or range (typically between 1:500 and 1:10,000 for primary antibodies). You’ll want to...from ascites fluid, try a dilution range from 1:1,000–1:100,000. Secondary antibodies For secondary antibodies...recommended dilution range is usually between 1:5,000 and 1:200,000. Check the manufacturer’s recommendations...antibodies that are not.   Table 1: Troubleshooting a western blot *indicates a diagnostic-only step Issue...reagent (usually an antibody).      Figure 1: A protein gradient. The density of the bands, which...

  5. Lambda Red: A Homologous Recombination-based Technique for Genetic Engineering

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    Blog Post
    ...recombineering system has three components (Figure 1): 1) Exo, 2) Beta, and 3) Gam. All three are required...that occur during DNA replication. There are two ways to avoid activating MMR: 1) use a strain of bacteria...Table 1: Overview of ds- and ss-DNA Uses in Recombineering Substrate Best Suited Application Recombination... interested in science communication and in vitro diagnostics.     References 1. Costantino N, Court DL...from the 5’ end and generate 2 possible products: 1) a partially dsDNA duplex with single-stranded 3’ ...recombineering with an ssDNA oligo substrate.   Figure 1: Components of the Lambda Red Recombineering system...and tags.  The typical frequency of recombinants is 1 positive clone out of 104 to 105 colonies. Pro Tip...

  6. Golden Gate Assembly Upgrades: More Fragments, Faster Assembly, and Higher Fidelity

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    Blog Post
    ... assembly reaction** 1 5 min., 37 ºC (2.5 μl) 687 100% 274,200 2,742,000 1 60 min., 37 ºC (2.5 μl)...References 1. Vladimir, Potapov, et al. "Optimization of Golden Gate assembly through application of ligation...expected complete sequence for the lacI/lacZ genes (1), while sequencing of white colonies showed a mixture...representative transformation plates obtained from 1-, 12- and 24-fragment assemblies of the lacI/lacZ ...lacZ cassette, and illustrates how the volume of the 1 ml outgrowth spread on each transformation plate can...fidelity studies conducted by Potapov et al. at NEB (1, 2). While T4 DNA Ligase, the mainstay of most biotechnological...over 90% for 24-fragment assemblies (Figure 3, Table 1). These improvements (5- to 12-fold increases in the...

  7. Typing CRISPR Systems

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    Blog Post
    ...   Class 1 versus class 2 There are two CRISPR classes: Class 1 and Class 2. Class 1 CRISPR systems...archaea, though Class 1 remains largely underutilized compared to Class 2. Class 1 Despite their incredible...optimal plasmids to harness the power of Class 1 systems. Class 1 CRISPR systems can be further broken down...and 17 variants. Figure 1: Overview of the CRISPR classification hierarchy. The hierarchy flows...effector complex after binding to a target. Class 1 is the most abundant of the two, comprising about ...incredible abundance, Class 1 CRISPR systems are less popular in the lab. This can be attributed to the multi-Cas...Type I CRISPR systems make up the majority of Class 1, and are the most common type of CRISPR overall. Type...

  8. Viral Vectors 101: Voltage Indicators

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    Blog Post
    ...depths (1 mm), important for certain applications such as in vivo tissue imaging.   Figure 1: Ace-mScarlet...Voltage indicators are voltmeters for cells! In this blog we will review how these indicators work, their...fluorescence or voltage indicators.   Viral Vectors and Voltage Indicators As previously mentioned...Enhanced genetically encoded voltage indicators advance their applications in neuroscience. Curr Opin Biomed...Cellular communication and function depend on potential energy. Cellular activities like action potentials...their general applications, and why viruses are essential to their use as biological tools. Why Use Voltage...Voltage Indicators? Electrical signals are integral to many facets of biology. For example, many heart...

  9. Antibodies 101: Normalization and Loading Controls for Western Blots

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    Blog Post
    ...pipetting or irregular membrane transfer. Figure 1: A loading control is a type of positive control necessary...because the loading control band is weaker in condition 1 than condition 2, the normalized values for the two...to using loading controls accurately. Assumption #1: The loading control is not perturbed by the experiment...without your knowledge? If you can’t trust Assumption #1, any change in the normalized intensity of your POI...interest band is significantly lighter in “condition 1” than “condition 2.” By eye, the loading control appears... the protein abundance axis, both blue “condition 1” points (POI and loading control) are smaller than...assumptions discussed above into question. Assumption #1: The loading control is not perturbed by the experiment...

  10. AAVs CREATed for Gene Delivery to the CNS and PNS

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    Blog Post
    ...nervous system (CNS). AAVs are popular tools because: 1) their genomes are easy to manipulate, 2) they have... stained for a neuronal marker). Check out tables 1 and 2 below for a summary of the quantified transduction... AAV vectors. What can these AAV’s be used for?  1. Multi-color labeling of neurons Multi-color labeling... dendrites extend to. The keys to this method are 1) high levels of color diversity so that cells are ...AAV.PHP.eB 69 +/- 4% 55 +/- 7% 76 +/- 5% Table 1: Transduction efficiency of AAV-PHP.B versus AAV-PHP.eB...expression into a two part system (figure 4d):  Part 1: A cocktail of three AAV vectors expressing RGB proteins...neuronal transduction efficiency, as shown in tables 1 and 2, with a cell-type specific promoter or enhancer...

  11. Viral Vectors 101: Producing Your rAAV

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    Blog Post
    ...Inactivation of AAV Serotype 1 Vectors. Human Gene Therapy Methods, 28(1), Article 1. https://doi.org/10.1089...Figure 1).    Figure 1: Overview of key steps of AAV production, iodixanol gradient purification, and ...repeat sequences. Human Gene Therapy Methods, 23(1), Article 1. https://doi.org/10.1089/hgtb.2011.034 Benyamini...in the CNS. Molecular Neurodegeneration, 15(1), Article 1. https://doi.org/10.1186/s13024-020-00361-z ...lentivirus, AAV does not possess the ability to replicate itself and depends on helper viruses like adenovirus...herpes simplex virus to facilitate efficient replication. The most common production system, however, ...RNA genes; and the pRepCap plasmid containing replication factors and serotype-specific capsid genes.  ...

  12. Who Gives a Tweet? 9 Facts About Scientists on Twitter

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    Blog Post
    ...2017. 1. Among scientists, historians have the most Twitter users. Biologists rank #8. Figure 1: Highly...that these results could be biased for two reasons: 1) they are based on the US Occupational Employment ...female to male = 0.629 (the closer this ratio is to 1, the closer males and females are to parity - the ... authorship in the United States (0.428). 4. The #1 (re)tweeted domain by scientists was Instagram Social...links to research that isn’t behind paywalls. 5. The #1 (re)tweeted science domain by scientists was Nature...mostly because of this article: “A systematic identification and analysis of scientists on Twitter.” This...from the United States Standard Occupational Classification System  and Wikipedia. 2. Social and computer...

  13. The Effect of COVID-19 on Liu Lab Plasmid Requests From Addgene Data

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    Blog Post
    ...we observed one complication in the China data – there is a period of 1 month, from 1/22/20–2/23/20, where...was around 33% of that observed between Jan. 1 and Mar. 1, 2020. Figure 2: A simple approximation...paper. Green and red lines depict the sum of mean +1 and -1 std for each paper, respectively. The fitted...plasmid requests correspond to publications of popular papers. Figure 1: Plasmid requests by date. ... from 5/22/14 to 5/19/20). Each paper has between 1–32 plasmids and a total of 2–2,590 requests. We received...China. However, it is evident that if we believe that 1/22/20–2/23/20 are the more accurate dates of lockdown...subfields.  Plasmid requests correlate with paper publication The dataset includes 11,426 plasmid requests ...

  14. MXS Chaining

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    Blog Post
    ...assembly. Journal of biological engineering. 2010;4(1):1. Pubmed PMID: 20205762. PubMed Central PMCID: PMC2822740...with minimal spectral overlap between them (Table 1). In each construct, 3 copies of the fluorescent protein... enabled labeling of particular structures (Table 1). Each construct was flanked with a CMV promoter (...construct was then introduced to HeLa cells (Figure 1). The resulting HeLa cells show robust labeling, with...fluorophore at the respective subcellular structure (Figure 1). Here, MXS Chaining gave researchers the ability ... all four constructs into one 15 kb insert. Table 1: MXS Constructs for Labeling Cellular Structures ...maxima Tethering partner Subcellular localization 1 TagBFP 399nm/ 456nm histone 2B (H2B) Chromatin 2...

  15. Recombinase-based State Machines Enable Order-dependent Logic in vivo

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    Blog Post
    ...RSM (See performance data from the indicated figures in our paper (1)) Input Plasmid Output Plasmid Fig... S9 of our paper (1)). We anticipate that, depending on the context and application, some inducible recombinase...recombinase-based state machines in living cells” (1), into a more straight-forward, concise explanation...an RSM is to sequence the register. In our paper (1), we allowed individual bacterial cells to form colonies...in the Materials and Methods section of our paper (1). Lastly, it is possible to build gene regulatory ... not. How might one implement RSMs? In our study (1), we implement RSMs in E. coli with two plasmids (...inputs to endogenous gene expression (2). Our paper (1) explains how to arrange attB-attP pairs on a register...

  16. Adenoviral Vector Production and Troubleshooting

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    Blog Post
    ...incubation as it will result in very low titer. Amplification (1-2 weeks) - The rAdV-S is used to infect more...increasing scale over the course of 1-2 weeks. Each round of amplification should result in a 10-100-fold ...repeats (ITR) present at both ends.   Figure 1: A) The structure of an adenovirus: the virion is ... 5 key steps: Construction of the rAdV plasmid (~1 week): The AdEasyTM system is the most popular method...Addgene’s Adenoviral Guide webpage, and references 1, 2.*Pro-Tip*: once the correct recombinant pAdV plasmid...transfected with the AdV plasmid construct from step #1 and allowed to stay in culture for up to 20 additional...CsCl are commercially available. Titer calculation (1 or 10 days) Physical titer (particle count, rAdV genomes...

  17. Live and Let Dye: Self-Labeling Protein Tags

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    Blog Post
    ...imaging of intracellular targets. Nature Communications, 7(1), Article 1. https://doi.org/10.1038/ncomms10778...by modular envelope design. Nature Communications, 14(1), Article 1. https://doi.org/10.1038/s41467-023...formin and capping protein. Nature Communications, 14(1), Article 1. https://doi.org/10.1038/s41467-023...molecules in vivo. Nature Biotechnology, 21(1), Article 1. https://doi.org/10.1038/nbt765 Lavis, L. (2016...Few Complicated Non-natural amino acids 1 Various Many Complicated   Applications Organic...fluorescent proteins, opening up new applications.   Figure 1: Live-cell labeling strategies. The...up linked to whatever molecule you choose (Figure 1). Just express the fusion protein and add a specific...

  18. Important Considerations When Using AAVs

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    Blog Post
    ...Viral Vector Blog Posts from Addgene 1. Location, location, location! What do buying real estate and using...important factors related to location. Location (1): What tissue? Location (2): Where in the tissue/which...vectors without specific facilities (Biosafety Level-1). Other possible tools, lentiviruses for example, ...suspected cases of insertional mutagenesis in humans (1) and AAVs are non-enveloped viruses, so they are resistant...the right serotype? Keep asking questions!  Table 1: AAV Serotypes and Tropism Experiment  Detection...the vicinity of the injection. Examining serotypes 1, 8 and 9, not only do they undergo retrograde and ...types of research may be enabled by the use of AAVs? 1) Combined functional and anatomical mapping of circuits...

  19. Antibody Validation for Flow Cytometry

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    Blog Post
    ...dye, respectively. WT and KO cells were mixed in a 1:1 ratio and fixed in 4% PFA and permeabilized in 0.1%...antibodies were diluted to 1 µg/mL except for 14558, which was used at 0.35 µg/mL (1/100). * = monoclonal antibody...through their HLDA workshops. Let’s use TIM-1 (Single pass type-1 membrane protein), which has been designated.... 400,000 cells were stained with the indicated Synaptotagmin-1 antibodies and corresponding Multi-rAb...specific antibody:target combination of interest (Figure 1). It can therefore be helpful to try all three when...to label an intracellular target.   Figure 1: HCT 116 WT and SYT1 KO cells were labelled with a...between the wild-type and the KO population (Figure 1), further optimization can be performed to both maximize...

  20. Lentiviral Vector Uses and Overview

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    Blog Post
    ...HIV-1 were transduced with a lentiviral vector containing an antisense sequence against the HIV-1 envelope...Trono and collaborators turned to the lentivirus HIV-1, well-known to infect non-dividing cells. The first...plasmids. The packaging plasmid carried a mutated HIV-1 provirus that couldn’t package itself due to a few...plasmid encoded the desired transgene flanked by HIV-1 long terminal repeats (LTRs) that facilitate viral... targeting and tracing cells in the brain (Figure 1). 2nd generation lentiviral packaging system The graphic...infectivity, is often used as a substitute for HIV-1 Env, which can only infect CD4+ cells. 3rd generation...lentivirus in the comments section below! References 1. In vivo gene delivery and stable transduction of ...

  21. X-CHIME: Context Dependent Germline Knockout in Immune Cells

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    Blog Post
    ...in the immune system. Nature Immunology, 25(1), Article 1. https://doi.org/10.1038/s41590-023-01689-6 ...screening of genes in the immune system. Nature Communications, 10(1), 1668. https://doi.org/10.1038/s41467-019... strains and lentiviral expression vectors (Table 1). The four systems Name Function Plasmid ... GFP (vex) Rosa26-FlpO-ERT2; H11-Cas9 Table 1: X-CHIME systems Proof of Concept These systems were...was lethal (LaFleur et al., 2024).   Figure 1: Schematic showing the X-CHIME approach. Figure from...

  22. Degrading DNA with Cascade-Cas3

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    Blog Post
    ...Cascade's complexity. Figure 1: Schematic of Class 1 CRISPR types. Class 1 includes type I with a Cas3 ...up type I, the most abundant type in the Class 1 category. These type Is can then be further broken into...unidirectional genome editing in human cells. Nature Communications, 10(1). https://doi.org/10.1038/s41467-019-13226...collection! A “complex” system Cas3 is part of the Class 1 CRISPR family, which means it requires multiple Cas...the brunt of the mechanical work for editing. Class 1 CRISPR systems make up the majority of CRISPR-based...CRISPR-based immune systems in bacteria and archaea. Class 1 is further broken down into different types, with ...antiviral defense). Cascade is brought to the target location using a gRNA and can then recruit Cas3, which ...

  23. The Breast Cancer Microenvironment: A Tumor’s Backstage Team

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    Blog Post
    ...enzyme (among others): caveolin 1, also known as CAV 1. Usually, caveolin 1 promotes the cell life cycle ...caveolin 1, there’s less malignancy because there’s less proliferation. However, the lack of caveolin 1 in ...believes that miRNA may decrease the expression of TAK 1, an enzyme that controls apoptosis and regulates transcription...Agnieszka K., et al. "An absence of stromal caveolin-1 expression predicts early tumor recurrence and poor... these stromal cell modifications, stromal cells are also constantly communicating with a tumor, proving... was contributed by Bárbara Pinho, a science communicator at the Portuguese science museum "Fábrica Centro...Proficient in transportation, exosomes are tools of communication and provide malignancy to the microenvironment...

  24. Viral Vectors 101: The Retroviral Lifecycle

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    ...can use Figure 1 to compare the genomes of these two family members.   Figure 1: Gamma-retrovirus...Retroviruses” is an umbrella term which encompasses 1.) the gamma-retroviruses — that have been developed...of retroviruses called the lentiviruses (like HIV-1) — that have been developed into lentiviral vectors...Academy of Sciences of the United States of America, 54(1), 137–144. https://doi.org/10.1073/PNAS.54.1.137. ...1997). Functional RT and IN incorporated into HIV-1 particles independently of the Gag/Pol precursor protein...RNA as template. Journal of Molecular Biology, 79(1), 163–183. https://doi.org/10.1016/0022-2836(73)90277...Aids Full-Length Human Immunodeficiency Virus Type 1 DNA Synthesis. Journal of Virology, 82(5), 2376. https...

  25. Mouse Modeling, Part 2: Breeding and Crossing Mice

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    Blog Post
    ...approximately 1 in 16 mice that harbor homozygous alleles at both of your genes of interest. About 1 in 4 mice...inheritance: 1. Genotype your parental mice In order to ensure that your genetic modification will be passed...In Part 1 of our mouse modeling blog series, we covered techniques that can be used to introduce genetic...mice in your experiment. As you’ve learned in Part 1, there are many types of genetically engineered mice...example of mice that are derived from crosses. In Part 1, we explained how genetic recombination in mice expressing...expressed, or inducible (this is explained more in Part 1 of this series). In any case, Cre is a dominant phenotype...genetic modifications into mouse embryos. But once you generate a growing colony of genetically engineered...

  26. When is a Monomer not a Monomer? The Top Three Ways Your Favorite Fluorescent Protein Oligomerizes in Cells

    Type
    Blog Post
    ...Figure 1).   Figure 1: Each problem with dimerizing fluorescent proteins is illustrated. 1) Membrane... in nature are prone to dimerization (i.e. EGFP) [1, 2] or even form obligate tetramers (i.e. DsRed) [...active state due to closure by FP dimerization.   1. Transmembrane fluorescent protein fusions Transmembrane...bright, non-physiological, and difficult to ignore [1, 2]. 2. Fusion to an obligate dimer or oligomer Several... For a more detailed discussion see [5]. Problems 1 and 2 depend on protein concentrations. An imperfect... distance runner, gardner, and cook.   References 1. Snapp, E.L., et al., Formation of stacked ER cisternae...problem for fusion proteins. One of the major applications of FPs is to visualize the localization, dynamics...

  27. Viral Vectors 101: Integrase-Deficient Lentiviral Vectors

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    ...and no viral progeny are produced (Figure 1).  Figure 1: Comparison of the life cycle of a lentiviral...2004). HIV-1 gene expression: lessons from provirus and non-integrated DNA. Retrovirology, 1, 13. https... cannot integrate it into the host genome (Figure 1). IDLVs were first discovered by researchers investigating...you might choose an IDLV over a lentiviral vector: 1. No risk of insertional mutagenesis Lentiviral vectors..., H. E. (1996). Human immunodeficiency virus type 1 integrase mutants retain in vitro integrase activity...Berry, C., Ecker, J. R., & Bushman, F. (2002). HIV-1 integration in the human genome favors active genes...https://doi.org/10.1186/1742-4690-1-13. PubMed PMID: 15219234 PubMed Central PMCID: PMC449739 Yew, C.T., ...

  28. Plasmids 101: Expressing shRNAs for RNA Interference

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    Blog Post
    ...vectors. BMC Biotechnology, 6(1). https://doi.org/10.1186/1472-6750-6-1 O’Brien, J., Hayder, H., Zayed...endogenous miRNA machinery for processing (Figure 1). The shRNA sequence is transcribed to produce hairpin... recruits other silencing proteins. Figure 1: Comparison of shRNA and miRNA processing and silencing...,"rgb":"rgb(0, 82, 169)","rgba":"rgba(0, 82, 169, 1)"},"button_font":{"size":19,"styles":{"font-family...:"rgb(255, 255, 255)","rgba":"rgba(255, 255, 255, 1)"}}{% endraw %}{% end_module_attribute %}{% module_attribute...RNA-induced silencing complex. Molecular Cell, 82(1), 30–43. https://doi.org/10.1016/j.molcel.2021.11.026...progress in miRNA biogenesis and decay. RNA Biology, 21(1), 36–43. https://doi.org/10.1080/15476286.2023.2288741...

  29. Addgene Depositors Get More Citations

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    Blog Post
    ...the journal of publication. Table 1 provides an example calculation of the weights. Table 1: Journal Weighting...amassed nearly 2,400 citations in its first four years (1). In addition to publishing in Science, Professor ...papers without plasmids deposited with Addgene (Figure 1). Even aggregated this much, you can see the 2013...(F) Weight (=F/E) (G) Weighted Citations (=C*G) 1 2010 3 Nature  3.3% 10.5%   3.2  9.6 2 2010...website at: http://www.neil-t.com/ .   References 1. Cong, Le, et al. "Multiplex genome engineering using...databases available, Web of Science. For each publication, Web of Science provides basic bibliographic ...Science, PLOS One, and PNAS. We then broke the publications from these journals into two groups: those containing...

  30. CRISPR-based Genome Editing Systems For Editing in E. coli

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    ... Then plasmids are cured from the cell. Steps 1-1 and 1-2 can be done simultaneously. Image from Li et...coli K-12 strains, 2 E. coli B strains, 1 E. coli W strain, and 1 Tatumella citrea, with a total of 70 sites... update the pCas/pTargetF system.   Figure 1: The editing process begins with the addition of the...updates involved the following changes. In pCas9, we 1) replaced the promoter of gRNA-pMB1 with PrhaB, 2)...biosynthesis, we constructed a strain library with 1-11 copies of integrated genes. We obtained a stable...far, we’ve used the MUCICAT system in two ways: In our initial paper, we used MUCICAT to optimize the gene...industrial strain. Not only is MUCICAT better, it is faster. Using MUCICAT only needs 2 newly constructed...
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