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...events by HDR can be generated more efficiently by (1) improving editing accuracy by preventing re-editing...specifically incorporate homo- or heterozygous mutations. 1. Increasing HDR accuracy with CRISPR/Cas-blocking ...targeted alleles when gene editing.   Figure 1: Techniques to improve editing efficiency.   But...provided by blocking mutations. Essentially, you:  Step 1 - Introduce the blocking mutation together with the...developing novel iPSC-based model systems.     References 1. Cong, Le, et al. "Multiplex genome engineering using...many cell types and often results in biallelic modifications. Most commonly, DSBs are repaired by the nonhomologous...

...), paCas9-1 induced indel mutations via NHEJ (frequency of 20.5%) and induced modifications by HDR (frequency...2 (CRY2) and calcium and integrin-binding protein 1 (CIB1) proteins. The goal of both groups was to create... the Sata lab is composed of two fusion proteins: 1) the genomic anchor - an inactive, dead Cas9 protein...combination. The optimized LACE system consisted of: 1) CIBN-dCas9-CIBN, where CIBN is the N-terminal fragment.... crassa) (Kawano et al., 2015). Nicknamed paCas9-1 and consisting of the fusion proteins N713-pMag and...fold-induction of Cas9 activity (16.4-fold). This paCas9-1 light-inducible system was able to recognize the same...background activity of the system by modifying paCas9-1 using nMagC714 instead of nMagHigh1-C714, generating...

...setting out of the lab (coffee, lunch or beer) and in 1:1 conversations. This creates a level of familiarity... other posts in our Science Mentors series: Part 1: "What Makes a Good Mentor?" and 6 More FAQs About...problems, develop general technical proficiency, communicate your work, maintain ethical standards and interact...websites listing alumi and it is easy to use publication records, university websites, ResearchGate and...

...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... sequencing are more definitive, but visual identification is a good place to start before taking on more...resources are limited or for bioart and teaching applications. Find a chromoprotein for your experiment! ...events: The plasmid carrying the desired genetic modification integrates into the genome (Figure 3). Here,...of Allelic-Exchange Vectors for the Scarless Modification of Proteobacterial Genomes. Applied and Environmental...

...vector at 1 x 1013 vg/mL titer), while AAV-PHP.S is typically used at a dose between 3 x 1011 and 1 x 1012...chosen capsid before using it in an application.     Figure 1: AAV toolkit for targeted gene delivery...capsid applications. For example, AAV-PHP.eB is typically administered at a total dose between 1 x 1011...rodents and broad tropism in primates. Nature Communications, 14(1), 3345. https://doi.org/10.1038/s41467-023...easy to produce in high titer, but higher doses (>1 x 1012 vg total in mice) may increase the chances ...decision tree for selecting a capsid, while Table 1 gives a brief overview of published descriptions of...Capsids page to learn more about that collection. Table 1: Systemic capsid plasmids available from Addgene ...

...Table 1.  Table 1: Antibody data repositories and search engines Website Targets Application Purpose...which allow you to search many different antibody catalogs at the same time.  If you're looking for an antibody... specificities, from target-specific ones to application-specific ones to anything goes. We've shared ...

...allowed for the identification of proteins associated with the IRF-1 locus. Figure 1: Schematic of...the promoter region of the transcription factor IRF-1. Immunoprecipitation, using an antibody targeting ...References Campbell, Amy E., et al. "NuRD and CAF-1-mediated silencing of the D4Z4 array is modulated ...affinity purification with an anti-Cas9 antibody. These different tag systems and affinity purification schemes...genomic alterations. These studies utilize a catalytically inactive form of Cas9 known as dCas9 (Jinek ...interest targeted by a gRNA. CRISPR-mediated purification of a specific genomic region In 2013, the Hodaka...steps (Fujita et al., 2013). CRISPR-mediated purification of a specific genomic region alleviates these...

... genome which are required for replication and transcription (Figure 1). All viral genes are removed and...interested in genome replication and transcription of negative strand RNA viruses.   References 1. Buchholz, Ursula...cells that stably express T7 (e.g. BSR T7/5 cells) (1). We hope that minigenomes will greatly enhance our...reverse genetics. Springer Berlin Heidelberg, 2004. 1-41. PubMed PMID:15298166. 3. Hoenen, Thomas, et al...Technical Tips). Minigenome replication and transcription In order to replicate the minigenome, the viral...transcription and replication strategies of Marburg virus and Ebola virus by using artificial replication systems...allowing researchers to study aspects of the EBOV replication cycle under BSL-2 conditions (4). What is a ...

...plasmids to produce AAV: 1) the RepCap plasmid which provides the AAV replication (rep) and capsid (cap)... teaching me about AAV production and purification! References 1. Aurnhammer, C., Haase, M., Muether, ...plasmid. This whole process takes ~3 hours to complete: 1 hours hands-on time and 2 hours for the qPCR run and... repeat sequences. Human gene therapy methods, 23 1, 18-28. PubMed PMID: 22428977. 2. Choi, V.W., Asokan...this AAV purification video! It didn’t exist when I did my first iodixanol gradient purification, but I ...In this post, I’ll share some AAV production, purification, and titration tips, while also summarizing ...cap) genes. AAV replication uses the host’s polymerase, but requires Rep proteins to process a double-stranded...

...Figure 1), an altered epegRNA delivered with an enhanced pU6cm promoter, viral replicon amplification of ...of cargo, and heat treatment.  Figure 1: Left: recombined prime editing tools PE2max-NC and ePEmax3...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...single-molecule biophysics Fast and responsive voltage indicator ONE-GO biosensors kit Antibodies for neurodegeneration...2024.12.20.629623.  A fast and responsive voltage indicator with enhanced sensitivity for unitary synaptic...and more responsive genetically-encoded voltage indicator (GEVI) than previous versions. ASAP5 can be imaged...

... 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...in line 4 of Figure 4. Since the loxP sites are located in intronic regions, this gene will still be expressed...

...10.1038/s41551-024-01227-1. doi:  https://doi.org/10.1038/s41551-024-01227-1   CHARM: A compact epigenetic...biotinylated SpyTag-SpyCatcher linkers and spacer proteins ending with a GFP nanobody (Figure 1). This enrichment...highly-visible beads on the cryo-EM grid.     Figure 1: MagIC-cryo-EM for sample enrichment and structure...single-particle analysis of PRC2 with RvLEAMshort (1:6 molar ratio) with 10 minutes of glutaraldehyde crosslinking...enrichment streamlines the purification and minimizes sample loss while also supporting imaging, making it...scFvs for other targets or could be used for applications beyond cryo-EM.   Find plasmids for MagIC-Cryo-EM...interact with the air-water interface (AWI) before vitrification, they can be completely denaturated (left) or...

...Fun fact: A 1 cm block of polymer can generate many meters of fibers!     Fig. 1 - Overview ... and brain neural circuits. Nature Biotechnology, 1–13. https://doi.org/10.1038/s41587-023-01833-5 Resources...channelrhodopsin 2 (ChR2), a blue light sensitive cation channel, fused to mCherry to the brain tissue. ... several months after implantation of fibers, indicating the system was indeed stable for continuous monitoring...interfaces with the entire body, so the obvious next applications are to expand the tool to different brain-organ...

...the Monoclonal Antibody Research Institute dedicated to: 1) developing new ways of generating antibodies... factors such as PAX7, PAX6, AP-2 alpha and ISLET-1. In addition, we distribute monoclonal antibodies ...target proteins of interest for labeling, quantification, purification, chromatin immunoprecipitation and more...Freedom: The Expansion of Data Sharing in Research Publications ...

...integration into CRISPR-induced DNA breaks. Nature Communications, 10(1). https://doi.org/10.1038/s41467-019-12449...NGS) to identify genes of interest. Figure 1: Overview of in vitro AAV CRISPR screens. Created ...certain experiments. AAV can be safely handled at BSL-1, while lentivirus requires a BSL-2 setup. This requires...diseases. Signal Transduction and Targeted Therapy, 9(1). https://doi.org/10.1038/s41392-024-01780-w Additional...This reduces the chance of integration at random locations that could cause deleterious effects. It is important...multi-plasmid system, where you have one AAV vector dedicated to expressing Cas9, while the other houses the...

...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...

... Eric Campeau 21474 pLenti CMV V5-LUC Blast (w567-1) Firefly CMV Lentiviral expression of firefly luciferase...bacteria to aquatic animals to insects. Luciferase catalyzes the oxidation of a substrate, leading to the emission...advantages and disadvantages depending on the application, and different luciferases require different ... course experiments, but is too dim for some applications. NanoLuc® is small (19 kDa), very bright, and... available, making it ideal for a variety of applications. To learn more and plan your experiment, read...OgNLuc ) fusions for BRET-based imaging and other applications. And see PalmGRET , a membrane-anchored version...

...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...