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...and Therapeutic Purposes. Annu Rev Virol. 2014 Nov;1(1):427-51. DOI: 10.1146/annurev-virology-031413-085355...personalized medicine. References and further reading 1. FDA approves gene therapy for blindness 2. Gene Therapy... Med Biol. 2014; 801: 687–693.  doi:  10.1007/978-1-4614-3209-8_86. PubMed PMID: 24664759. PubMed Central... time advances in human genetics led to the identification of several disease-causing genes, including... But it is the hard work and persistence of a dedicated team of scientists led by Dr. Jean Bennett (UPenn...-1250015778 7. Yu W and Wu Z., 2018. In vivo Applications of CRISPR-Based Genome Editing in the Retina...

...DNA with a PCR purification kit or with a phenol-chloroform extraction.    Fig. 1: Schematic overview...reserve a small aliquot, split into two parts to (1) run a DNA gel to confirm fragment size and (2)  incubate... let’s do a quick review. At this point you have (1) crosslinked your proteins of interest to any DNA ...next-gen sequencing. ChIP is an incredibly useful application for exploring these interactions, and can give... protein interactions, including epigenetic modifications. But it's not an approach for the faint of heart...background information. Your options for this step are sonication, which uses high-frequency sound waves to break...break up the DNA, or enzymatic digestion. Sonication provides a truly random digestion, but is harsher ...

...Time: Topic) 0:00-1:00 : Intro 1:01-3:48 : Kingdom Kwapata's background and education 3:49-4:35 : Kingdom...Born to a family in Malawi that pushed science education, Kingdom Kwapata grew up to be one of the best...

...overlap with the negative control population (Figure 1). Based on where the GFP- population ends, you can...total cells present in the gate.   Figure 1: Example of yes-no flow plot of the (negative) control...for a yes-no dot plot, like the one shown in Figure 1.  Figure 3: An example of a histogram plot ...to the right indicates a higher signal, or upregulation, and a peak to the left indicates a lower signal...anything to the right of it, marking it as GFP+. Quantification for such graphs could simply be the percentage...case for you, switching to a contour plot should indicate where the two populations diverge. The smear ...place your gate based on the dot plot would be complicated.  Figure 2: (A) a smeary dot plot showing...

...the viral TF library, with each cell receiving only 1 TF expression construct. Cells were cultured following...states was generated (Joung et al., 2023). Fig. 1: Barcoded TF library was packaged in lentiviral particles...atlas of directed differentiation. Cell. 2023. 186 (1) 209-229. doi: 10.1016/j.cell.2022.11.026 Joung J....unique barcodes for each TF help streamline identification. While the Zhang lab controlled their multiplicity...single TF was delivered to each cell, future applications could deliver multiple TFs to each cell, allowing...processes. For help with library transformation and amplification, refer to the Zhang lab’s previously published...published protocol and its relevant modifications on the pooled library webpage. Disease models Good disease...

... skills in science communication will be a positive asset throughout your career? 1. Make scicomm practice... the Communicating Science Conference series for graduate students. I believe that communication is the...skill in science communication over time.  Like most skills, great science communication comes with practice...able to communicate with their colleagues and peers. Scientists also need to be able to communicate to expert...need to communicate with non-scientists, to establish broader impacts for their work in education and policy...with opportunities to practice communicating.  Some fantastic K12 education initiatives like BiteScis (https...careers in science communication The strength of story telling Science communication: 9 strategies to get...

...give your research more visibility.    Figure 1: Illustration of SARS-CoV-2 from Philippa Steinberg...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...other proteins that require post-translational modification may not be easily expressed in non-mammalian...constructs contain protein coding genes, tags, truncations, and codon optimizations similar to those found...are intended for the high level expression and purification of SARS-CoV-2 proteins for downstream experiments...wild type and codon optimized, full length and truncated versions of SARS-CoV-2 S (spike protein) cloned...

...plasmid. This phenomenon indicates the presence of a multimer.   Fig. 1: The monomer of Plasmid...and catenanes by agarose gel electrophoresis. Methods Mol Biol. 2009;582:11-25. doi: 10.1007/978-1-60761...plasmid sequencing services using long-read sequencing (1-25 kb), such as Oxford Nanopore sequencing or PacBio...10017-26. doi: 10.1074/jbc.M110.176636. Epub 2011 Feb 1. PMID: 21285356; PMCID: PMC3060453. Bazaral M, Helinski...multiple circular DNA forms of colicinogenic factor E-1 from Proteus mirabilis. Biochemistry. 1968 Oct;7(10...additional origins of replication compared to monomers, they are also able to replicate at a higher frequency...the right, with the entire monomeric sequence duplicated in tandem, resulting in a plasmid that is twice...

...between 2 loxp sites and is excised by Cre (Figure 1). This results in one loxp site and one lox2272 site...cause of this spurious expression?   Figure 1.  Cre-recombinase binds to either pair of lox sites...recombination and can exhibit recombination levels as high as 1-3%. In light of this finding, we now include guidance...Efficiency, Tropism and Axonal Transport of AAV Serotypes 1, 2, 5, 6, 8 and 9 in the Mouse Brain. PLoS ONE 8:e76310...Vectors Pseudotyped with Viral Capsids from Serotypes 1, 2, and 5 Display Differential Efficiency and Cell...promoter, such as the truncated tyrosine hydroxylase (TH) promoter for catecholamine neurons or glial fibrillary... Flp), to either flip or excise a gene that is located between them. Early recombinase-dependent vector...

...or nuclear expression of BFP2.     Figure 1: CROPseq-multi uses two sgRNAs with internal barcodes...transcript by endogenous processing enzymes (Figure 1). To ensure this tRNA processing only occurs on the...served as positive control (cis-ctrl). v1, vector 1; v2, vector 2; BD, binding domain; SDS, splice donor...)genome editing and gene therapy. Nat Commun., 14(1), 6578. https://doi.org/10.1038/s41467-023-42386-0...mice. B) Quantifications showing high microglia specificity of GFP expression for the indicated ssAAV5s...ssAAV5s. C) Quantifications showing high microglia transduction efficiency for the indicated ssAAV5s. Figure...3’ long terminal repeats (LTR). The 3’ LTR is duplicated during lentiviral integration, producing a second...

...that (1) the sequence is unique compared to the rest of the genome and (2) the sequence is located just...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, ...editing is most efficient when target sites are located in close proximity to the region to be edited. ... that were able to cleave target DNA sequences located upstream of either NGA or NGC PAM sequences (Kleinstiver...variant may increase the specificity of genome modifications at DNA targets adjacent to NGG PAM sequences...

...the whole story Figure 1: Schematic representations of the indicated unbiased genotyping options.... and reproducibility of a study’s findings. Table 1: Biased Genotyping Options Technique Sequence Information...types Must be verified with cell-based method IDLV 1% Cell-based Programmable, can detect DSBs in live ...magazine.scienceforthepeople.org/.     References 1. Fu, Yanfang, et al. "High-frequency off-target mutagenesis...next-generation sequencing." BMC genomics 15.1 (2014): 1. PubMed PMID: 25409780. PubMed Central PMCID: PMC4246457...off-target sites across the genome and pinpoint the location of mismatches based on the sequences of the genome...those relating to DNA structure (e.g. epigenetic modification, bulges) that may also impact off-target edits...

... dual-color control of neurons. Nature Communications, 12(1), 1–20. https://doi.org/10.1038/s41467-021...to consider when choosing one.     Figure 1: Example of an experimental optogenetic setup. A light... 21–51). Springer US. https://doi.org/10.1007/978-1-0716-0755-8_2 Lehtinen, K., Nokia, M. S., & Takala... 21–51). Springer US. https://doi.org/10.1007/978-1-0716-0755-8_2 Boyden, E. S., Zhang, F., Bamberg, E...channelrhodopsin-2 (ChR2), a blue light-activated cation channel, from the archaebacteria Chlamydomonas ...stimulation. GPCR signaling GPCR signaling is a complicated process involving many downstream signaling mechanisms...break your experiments!  First, closely read the publication, including the supplemental data. You’re most...

... is conjugated to a fluorophore (Figure 1).   Figure 1: Comparison between how direct immunofluorescence...important considerations are to make sure your secondary 1) will recognize the species of your primary antibody...New York, pp 299–311. https://doi.org/10.1007/978-1-4939-8935-5_26 Stuurman N. (2012) Introduction to ...knowing that where you see fluorescence indicates your target’s location. Some other terms you might see associated...immunofluorescence? Generally speaking, IF is an application in which antibodies that have been joined to ...bind to a single primary, resulting in signal amplification. Another reason is cost effectiveness. Generating...distribute antibodies validate them in a variety of applications, including IF (or IHC or ICC), and will share...

... the samples stored in our lentivirus tubes (Fig. 1). However, once thawed, there was a substantial reduction...in the pH of samples stored in our AAV tubes (Fig. 1). It’s important to note that the change in pH only...using the thawed virus within 24 hours. References 1. Murphy BM, Swarts S, Mueller BM, van der Geer P, ...improve our shipment processes. After reading a publication describing how short term storage on dry ice ...medium, which is buffered and contains the pH indicator phenol red. We froze samples in a -80°C freezer...closed dry ice storage container for 48 hours (to replicate how our viruses are shipped), where they would...exposed to CO2/dry ice. All samples were tested in duplicate. What we found Not all tubes (or rather, not ...

...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. ...enough to ensure the virus can replicate and spread. Instead, viruses replicate by hijacking the host’s system...low immunogenicity which makes them ideal for applications with whole organisms. Finally, viral delivery...delivery is typically straightforward across applications, while electroporation of a human, for example, isn...people in nature, extra safety measures, like replication incompetent viral particles, are required. We...

...include mEGFP, mCherry, and HaloTag. Figure 1: (a) Anti-FLAG frankenbody (FB) construction from ...Comm. 2019. https://doi.org/10.1038/s41467-019-10846-1.   Optimized genetic screening technique using Cas12a... to Cas9’s larger expression requirements (Figure 1), but Cas12a has generally been underutilized in genetic...editing applications.  Figure 4: Overview of CasMINI for genome engineering applications. Image ...stability in cells, and use it in a variety of applications. This set of new anti-FLAG frankebodies includes...to FLAG-tag H2B for nuclear localization. (c) Applications of anti-FLAG-FB-GFP labeling various targets... and anti-HA frankenbodies have a variety of applications, including live-cell imaging, single-molecule...

...available from their original sources. Figure 1: Biological repositories that distribute mouse strains... from Reproducibility for Everyone. Case study 1: Cell lines Many of the reagents used to investigate...COVID-19: an observational study. The Lancet Microbe 1:e14–e23 . https://doi.org/10.1016/s2666-5247(20)30004...tropism, replication kinetics, and cell damage profiling of SARS-CoV-2 and SARS-CoV with implications for ... Another example of reagents that have broad applications but have proven useful to the study of SARS-...vectors VSV-G and psPAX2, which were used in a publication by Xiuyuan Ou, et al. that characterized the ... requested these plasmids from Addgene. This publication then resulted in the development of a new plasmid...

...s pLKO.1 protocol. Pro-tip: this plasmid grows more slowly than standard plasmids. Find pLKO.1 - TRC cloning...experiments.  Find the scrambled shRNA vector. pLKO.1 - TRC cloning vector - This vector from the David ...taken us on many adventures...From moving office locations three times, to opening our UK office in 2014,...cell types so it is useful in a wide range of applications. This plasmid was the top requested plasmid ...notice the “V2.0” at the end of this plasmid which indicates that there was a prior version of this plasmid...plasmid was called pLKO-Tet-On in the original publication but its name was subsequently changed to clarify...

...and CasPEDIA have got you covered!     Fig. 1 – CasPEDIA Cas ID display information for a given ...established uses, and intended applications. Within CasPEDIA, there is a classification system, CasID, to help...multiplex ability Experimental guidelines and applications Protein structure and properties Links to ...you want to read literature on the less common applications of an enzyme for your non-traditional experiment...al. (2023). CasPEDIA Database: A Functional Classification System for Class 2 CRISPR-Cas Enzymes [Preprint...I., Iranzo, J., et al. (2019). Evolutionary classification of CRISPR–Cas systems: a burst of class 2 and...