...course much more difficult than in cell culture, but Ran et al. have shown that CRISPR and AAV can be used...Thakore PI, Moreb EA, Rivera RM, Madhavan S, Pan X, Ran FA, Yan WX, Asokan A, Zhang F, Duan D, Gersbach CA...WL, Widrick JJ, Yan WX, Maesner C, Wu EY, Xiao R, Ran FA, Cong L, Zhang F, Vandenberghe LH, Church GM, ...: 25692716. PubMed Central PMCID: PMC4335351. 9. Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz...X-linked recessive disorder affecting approximately 1 in 5000 males. DMD is caused primarily by frameshift... applicable to 80% of DMD patients. Figure 1: Exon skipping strategies for Duchenne muscular dystrophy...needed. First, CRISPR delivery must be optimized to: 1. Reach a high percentage of muscle cells throughout...

...obstacles when working with virus: 1. Viral vectors undergo DNA rearrangements. Depending on the composition...approximately 20–50% of the packaging cells after 1–3 days of selection. A lower percentage of surviving...up-to-date tips for your virus experiments. References 1. Anthony, Simon J., et al. "A strategy to estimate...may undergo genomic rearrangements during replication. To avoid genomic rearrangements, try amplifying the...immediately prior to their transduction has been shown to increase AAV transduction efficiency by approximately...immediately following transfection. In other words, when exchanging the medium after transfecting cells, replace...Fibronectin is another membrane-interacting protein that can be used to enhance transduction efficiency in cells...

...if you obtained a transformant with a single insertion because it will segregate 3:1 in the T2 generation...lineage and has made countless transgenic plants in the process.     References 1. Emami, S., Yee, M. and Dinneny...Methods. BioMed Central, 2(1), p. 19. doi: 10.1186/1746-4811-2-19. PubMed PMID: 17087829. PubMed Central... which is ease of transformation. There are many motivations to generate transgenic Arabidopsis, from ...from studying transcriptional and translational dynamics of genes and proteins in living plants, to complementing... dip transformation method. The general steps for this method include: Cloning and transforming a plasmid...integrates transfer DNA (tDNA) into the genomes of the plants it attacks Growing the transformed agrobacterium...

...focus on the AAV transfer plasmid and take a look at each of its parts. Figure 1: Parts of an AAV...human synapsin 1 promoter is neuronal specific (Haery et al., 2019). Most AAV transfer plasmids have a...repeats (ITRs) are what makes an AAV transfer plasmid an AAV transfer plasmid. ITR sequences are 145 bases...read through this challenging sequence. Transgene The transgene is the gene that will be delivered by the...control transgene expression. Lox sites are directional 34 bp sequences that can flank the transgene and ...(WHP) Posttranscriptional Regulatory Element (WPRE) forms a tertiary structure when transcribed which ... nuclear export of RNA and RNA translation, and promotes RNA transcript longevity. The polyA is located...

...Check out Addgene's New Viral Service! References 1. Hay, R.J., Macy, M.L. & Chen, T.R. “Mycoplasma infection...contaminations.” Methods in Molecular Biology 946 (2013). 1-13. Pubmed PMID: 23179822. 5. Segura, M.M., Kamen,...Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors."Nature protocols 4.4 (2009...may not translate well to other cell types. To overcome this issue, try transducing with a range of dilutions...DEAE-dextran can improve transduction efficiency. In addition, this study found that transduction efficiency...passage number of a culture the lower the transfection and transduction efficiencies. Cultures should only be...others opt for a single harvest after transfecting with the transfer and packaging plasmids. A benefit of...

...PDF 2.3 MB Zhang GeCKO pooled library amplification 1 vector system: lentiCRISPR v2 2 vector system: lentiCas9...and Wente Zebrafish: gRNA cloning, in vitro transcription, injection gRNA core ; Cas9 ; optimized Cas9...102.5 KB Jaenisch CRISPR plasmid summary for transcriptional activation dCas9 activators sgRNA dual expression...

...regulated in the medicinal plant, the Catharanthus roseus (Figure 1). The ultimate goal of our research ...reports 9.1 (1990): 1-5. PubMed PMID: 24226366. Lee, Min Woo, and Yinong Yang. "Transient expression assay... plant scientists do transient leaf transformations The development of transgenic plants is a common problem...that the transformation and gene expression can be tracked. During a transient leaf transformation, the engineered...method. Transfer your plant into the dark 16 hours before the transformation. We routinely transfer our plants...developing transgenic hairy root lines in tissue cultures takes half a year, and generating a transgenic plant...plant can take even longer, a transient plant leaf transformation process could save the plant biologist...

...glossary at the end of this post! Figure 1: Base transitions are edits between the purine bases (adenine...uracil DNA glycosylase. Nature Communications, 15(1), 4897. https://doi.org/10.1038/s41467-024-49343-5...uracil-DNA glycosylase. Nature Communications, 15(1), 6397. https://doi.org/10.1038/s41467-024-50012-w... the non-edited strand to encourage the cell to repair the DNA using the edited strand as a template —...editing. Like other transversion editors, this editor had relatively high indel rates ranging from 4.7% to 30%...our last post, we talked about the first base transversion editors: CGBEs, or C → G Base Editors. CGBEs...editor could excise other bases? Could other transversion editors be close at hand? Once again, this post...

...overcomes many of those limitations (Figure 1). Figure 1: Opto-katanin induces fast, localized microtubule...Signaling Toolbox. ACS Synthetic Biology, 11(1), 515–521. https://doi.org/10.1021/acssynbio.1c00604.  Mahlandt...specific area at the plasma membrane (Figure 2). Just express the plasma membrane-localized iLID along with...cell membrane isn’t powerful enough, what about altering the chemical composition of the membrane itself...SuperPLD hydrolyzes membrane PC to PA. A) OptoPLD is recruited to plasma membrane or lysosomes and its...optogenetic tools to control the cytoskeleton and cell membrane dynamics. First, a quick refresher.  The tools...undergoes a conformational change and binds the plasma membrane under blue light CRY2-CIB1 heterodimerizes or ...

...mediating the entry of SARS-CoV and SARS-CoV-2 viruses. ( 1 ) TMPRSS2 - a serine protease that primes the SARS-CoV... Plasmids expressing Cas12a/Cas13a, Reverse Transcriptases, etc. that may be useful for detection assays...site for furin proteases.( 3 ) BSG - (CD147), transmembrane glycoprotein of the immunoglobulin superfamily...proteolysis of SARS-CoV Spike protein, activating its membrane fusion function.( 5 ) TACE - (ADAM17) a metalloprotease... method termed DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR). Mammoth Biosciences has published...2020.06.23.166397 (Link opens in a new window) reverse transcriptase, protease, and Bst polymerase constructs now...unpublished) Taq polymerase and MMLV reverse transcriptase expression plasmids from the Drew Endy and Philippa...

...whether they could 1) be decorated with proteins and remain functional, 2) break membranes outside of their...Follow her on Twitter @jessicapolka.     References 1. Kanabrocki, J. A., Quackenbush, R. L. & Pond, F. ...learn a lot from Nature’s simpler solutions to transmitting forces over long distances. For instance, how..., R bodies do not exert force by dynamically rearranging into different polymerization states. Instead...demonstrates that R bodies can function to break membranes of different organisms. And finally, we demonstrated...could use them to lyse cells (and surrounding membranes) in keeping with their natural function (see below...

...genome upon viral transduction. Many lentiviral transfer plasmids are based on the HIV-1 virus. For safety... the transfer plasmid must be modified. Most shRNA‐expressing lentiviral plasmids, such as pLKO.1, use...protocol for using the popular cloning vector pLKO.1. Trono Lab Resources: Lentivectors Toolbox and Maps...selection. Desfarges S, Ciuffi A. Viruses. 2010. Jan;2(1):111-30. PubMed . Altering the tropism of lentiviral...Curr Gene Ther. 2005. 5(4): 387-398. PubMed . HIV-1 Genome Nuclear Import Is Mediated by a Central DNA... can be used in transient transfections to achieve expression of the transgene, and those that can are.... Lentiviral transfer plasmids are not designed specifically for transient transfections. Therefore, there...

...plasma HIV-1, improving life expectancy and quality for HIV-1 patients. However, ART is not an HIV-1 cure. ...activated HIV-1, measured via HIV-1 promoter-driven GFP expression. In cell lines that can produce HIV-1 toxic...with HIV-1. For two HIV-1 strains, Cas9/gRNA lentiviral expression significantly reduced the HIV-1 copy number...HIV-1 transcription and destroy infected cells, the other using wild-type Cas9 to remove the HIV-1 genome...currently infected with the lentivirus HIV-1. Today, HIV-1 can be controlled with antiviral therapies ...take on HIV-1 in vitro, and what obstacles must be overcome for clinical success. ART and HIV-1 reservoirs...Cutting HIV-1 out of the genome with CRISPR/Cas9 Before the introduction of CRISPR, HIV-1 genome editing...

...Addgene, we use seven different types of “plates” (Fig. 1). When you have to tell an engineer that you want ...”? We took steps towards solving this problem by 1) making sure we qualified these words when we discussed...resources on the Addgene blog Read about other transferable skills Find all of our science career related...

...offers insight into a career as a Professor. Step 1: Your PI forwards you a Request For Applications (...proposal typically lists the deliverables, explains 1-2 potential pitfalls of the intended protocol, and...improve your grant writing skills. Good luck! Further advice on grant writing Since successful grant writing... during the grant application process. When my PhD advisor first asked me to write a grant application... advice on writing a grant proposal. "Murder Most Foul: How Not to Kill a Grant Application" by Vid Mohan-Ram...their advisor alone applies for these competitive grant applications; however, in some cases, your advisor...advisor may choose to allot some if not all grant duties to trusted students. For example, if the proposal...

...   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...6105. https://doi.org/10.1093/nar/gku241 Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N....types, 33 subtypes, and 17 variants. Figure 1: Overview of the CRISPR classification hierarchy. ...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...

...Communications , 15 (1), 3663. PMID: 38688943 Hsu, P. D., Scott, D. A., Weinstein, J. A., Ran, F. A., Konermann...pooled format into lentiviral transfer vectors (Figure 8B). Libraries come in 1-vector systems, in which Cas9..., 1163–1171. PMID: 23979020 Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D..., 17 (5), 471–479. PMID: 32203383 Nishimasu, H., Ran, F. A., Hsu, P. D., Konermann, S., Shehata, S. I....CRISPR-Cas9. Cell , 167 (7), 1829-1838.e9. PMID: 27984730 Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott...Nature Protocols , 8 (11), 2281–2308. PMID: 24157548 Ran, F. A., Cong, L., Yan, W. X., Scott, D. A., Gootenberg...Communications , 14 (1), 6175. PMID: 37794046 Small precision edits Anzalone, A. V., Randolph, P. B., Davis,...

...improve your grant writing and hopefully increase your success rate. 1. Submit your grant early! First...involved so it’s important not to throw in the towel if 1 or 2 funding bodies say no. If possible, get feedback...in writing small travel grants to attend conferences and a seed funding grant for a proof-of-principal...www.assaygenie.com. No matter what facet of academia you are in, grant writing can be the lynch pin that results in your...to be much use. 2. Originality is essential for grant success The two most important things reviewers ...sure that comes across. Check out some in-depth grant writing tips at the Human Frontier Science Program's...Skills 5. Practice makes perfect, apply for small grants too There are actually quite a lot of different...

... phototropin 1 LOVETRAP reversibly sequester and release proteins from cellular membranes using light....algae Chlamydomonas reinhardtii . Channelrhodopsin-1 (ChR1) is excited by blue light and permits nonspecific...concept of optogenetics. 2012 Prog Brain Res. 196: 1-28. PMID 22341318 Gradinaru V, Zhang F, Ramakrishnan...diversifying and extending optogenetics. Cell. 196:1-28. PMID 20303157 Han X, Boyden ES. 2007 Multiple-...Limitations and Future Developments. Exp Physiol. 96(1): 19–25. PMID 20621963 Mattis J, Tye KM, Ferenczi ...wavelengths, ranging from blue to yellow to red. Red light exhibits better tissue penetrance, which may ...including protein activation, membrane localization, and transcriptional activation. In the widely used...

...and placed in a buffer. Antibody Structure Figure 1: Structure of an antibody A standard antibody is made... together to form a “Y” shape, as shown in Figure 1. The two arms of the Y structure are responsible for...proteins by size. Transfer proteins from the gel to a membrane. Incubate the membrane with an unconjugated...block the membrane to remove excess antibody and prevent unwanted binding. Incubate the membrane with a ...Wash and block the membrane. Activate the HRP with a substrate kit. Image the membrane using either X-ray...hybridoma cells into plasmids. Plasmids are then transfected into cells and induced to express antibodies....variable regions, shown in light blue and light orange, that bind to a specific antigen, triggering an...