... than control, 3; same as control (Cetn1/sgRNA1), 2; darker than control, 1; very dark). Brighter fluorescence...you can hold off on genotyping until the pups are 2-4 weeks old and use the ear tag method (creating different...PMID: 24253446. PubMed Central PMCID: PMC3875854. 2. Mashiko, Daisuke, et al. "Generation of mutant mice...target sequence was digested by sgRNA guided Cas9 endonuclease, homology dependent repair (HR, homologous recombination...off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases." Genome research 24.1 (2014): ...

...required components are 1) a SMASh-tagged protein and 2) asunaprevir (small molecule). dTAG The degradation...gone through an additional round of evolution (AID-2) to reduce some of the leakiness associated with the...become increasingly popular for the modulation of endogenously and exogenously expressed proteins. Here we ...Secondly, these tags can be used to manipulate endogenously and exogenously expressed proteins. Thirdly,...dimerization of the FKPB12F36V fusion protein with the endogenous CRBN E3 ligase complex which will polyubiquitinate...the dTAG uses a small molecule, a tag, and the endogenous cellular degradation machinery to function. The...The TIR1 F-box protein will associate with the endogenously expressed SCF complex members, an E3 ubiquitin...

...are many docking sites available for integration. 2. How soon would you like your flies ready for use?...beautiful immunofluorescent images like in this paper (2). Metabolic assays such as Seahorse can be employed...PMID: 16547094. PubMed Central PMCID: PMC1526508. 2. H. Jiang,  et al. Cytokine/Jak/Stat signaling mediates...which restores eye color to fly lines where the endogenous white gene is knocked out. Flies with your integration...

...conjugation. 1) Endogenous lysine residues, which can be found throughout the antibody. 2) Endogenous cysteines...Detecting antigens by quantitative immuno-PCR. Nat Protoc 2:1918–1930. https://doi.org/10.1038/nprot.2007.267 ...the disulfide bonds between antibody chains. 3) Endogenous carbohydrates added to antibodies post-translationally...

...Currently available from the Kim lab are 2 presynaptic and 2 postsynaptic targeting mGRASP plasmids. Additionally... cells were pulsed with 2µg/ml of Doxycycline for 2 hours, and imaged on the Keyence BZ-9000 fluorescence...work! Robinson et al., Dev Cell 2010 Feb 16;18(2):324-31. Cheeseman et al., J Cell Sci 2013 May 1...promotes the formation of an isopeptide bond between 2 peptide tags, SpyTag and KTag. The group demonstrated... of three sets of cloning vectors (Level 0, 1, or 2) which can be utilized in three successive assembly...1 modules can be similarly assembled into a Level 2 vector, thus forming a functional genetic circuit....circuit. Flexibility has been built into the Level 2 vectors to allow for additional iterations of Level 1 assembly...

...proteoglycan (HSPG)3 AAV receptor (not strictly required)2 Human fibroblast growth factor receptor 1 (FGFR1... AAV2, so they are formally called rAAV2/1, rAAV2/2, and so on, although we often use abbreviated nomenclature...suggest these serotypes as a good place to start. Table 2: AAV tissue tropism for mice in vivo. To target... tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PloS One, 8(9),...Retrograde Access to Projection Neurons. Neuron, 92(2), 372–382. https://doi.org/10.1016/j.neuron.2016.09.021...reported to have tropism for lung, liver, vascular endothelial, and smooth muscle cells (Issa et al., 2023)....

...CombiGEM lentiviral vector backbone pAWp12 (Figure 2). Cloning from the pAWp28 storage vector to the pAWp12... before beginning your experiments. Figure 2: Strategy for assembling barcoded combinatorial gRNA...PMID: 25114216. PubMed Central PMCID: PMC4151723. 2. Doench, John G., et al. "Optimized sgRNA design to...targeting your genes of interest. Thanks to the tremendous efforts made by various research teams to build...

... (2,3). Stable strand separation is maintained by 2 sets of interactions : 1. Interactions between the...formed by the Cas9 HNH and RuvC nuclease domains 2. Interactions between the target strand and the gRNA... Find plasmids from this publication at Addgene. 2. Nishimasu, Hiroshi, et al. "Crystal structure of ...PAM-dependent target DNA recognition by the Cas9 endonuclease." Nature 513.7519 (2014): 569-573. PubMed PMID...

...calcium biosensor, so that the (1) binding of calcium, (2) allosteric rearrangements of the sensor, and (3) ...user-controlled illumination with violet light and (2) exposure to increased intracellular calcium. It has...dynamics in vivo. Neuroscience Research. 2021 Aug 1;169:2-8. Suzuki J, Kanemaru K, Iino M. Genetically encoded... prevent aberrant binding of the biosensor to endogenous calmodulin. Multiplexing Are you combining your...

...Nature Cell Biology, 26(2), 294–304. https://doi.org/10.1038/s41556-023-01342-2 Koblan, L. W., Arbab, M...order to bind DNA, Cas9 requires (1) a guide RNA, (2) a complementary DNA sequence, and (3) a protospacer-adjacent...region of separate strands called an R-loop (Figure 2). This allows the guide RNA to base pair with the ...accessible on the outside of the enzyme. Figure 2: CRISPR R-loop. Created with BioRender.com. Base...), 35–40. https://doi.org/10.1038/s41587-020-0592-2 Additional Resources on the Addgene blog CRISPR 101.... They conducted a CRISPRi screen to identify endogenous genes that impact CGBE efficiency. In addition...

...revert back to a pluripotent stage (iPSC formation) [2, 3]. The iPSCs then proliferate and redifferentiate... more common delivery methods include retrovirus [2], lentivirus [4], adenovirus [5], Sendai virus [6]...2006. 441(7097): p. 1061-7. PubMed PMID: 16810240. 2. Takahashi, K., et al., Induction of pluripotent stem... Pluripotent Stem Cells. Cell Stem Cell, 2015. 17(2): p. 178-94. PubMed PMID: 26189426. 15. Si-Tayeb, ...induced pluripotent stem cells. Stem Cell Rev, 2015. 11(2): p. 242-53. PubMed PMID: 25578634. PubMed Central... to regeneration. Nat Rev Mol Cell Biol, 2011. 12(2): p. 79-89. PubMed PMID: 21252997. 37. Merrell, A..... Patsch, C., et al., Generation of vascular endothelial and smooth muscle cells from human pluripotent...

... fluorophore excitation by absorption of a photon 2. Energy transfer from the excited donor directly to...PMID: 24194740. PubMed Central PMCID: PMC3810607. 2. Bajar, Bryce T., et al. "A Guide to Fluorescent Protein...PubMed PMID: 10500161. PubMed Central PMCID: PMC18018. 2. Miyawaki, Atsushi, et al. "Fluorescent indicators...biomolecules that are not proteins) or would perturb the endogenous state that you want to measure (overexpressing...

...AAVs in the clinic Thus far, AAV vectors AAV1, AAV-2, AAV1-AAV2 hybrids, AAV-6, AAV-7, AAV-8, AAV-9 and...neutralizing antibodies against some AAV types, AAV1, 2, 3 and 5 are thought to be as high as 70% among the... 10.24 (2003): 1999-2004. PubMed PMID: 14566358.  2. Hirsch, M. L., et al. "Self-complementary AAV mediates...mediates gene targeting and enhances endonuclease delivery for double-strand break repair." Gene therapy...

...super-resolution or single-molecule techniques.   Figure 2: Two-color super-resolution imaging of ER and mitochondria...stimulated emission depletion (STED) nanoscopy. Scale bar, 2 μm. Inset shows comparison with standard confocal ...enables applications like multi-color imaging (Figure 2). If you’re working with purified proteins in vitro...Labeling in Living Cells. Chemistry & Biology, 15(2), 128–136. https://doi.org/10.1016/j.chembiol.2008.01.007...conjugates: A flexible chemical tag. Nature Methods, 2(4), Article 4. https://doi.org/10.1038/nmeth749 Strebinger...for your experiment. Conclusion Researchers and vendors continue to develop new ligands for these systems...

...engineering methods have their own pros and cons (2). The NgAgo DNA guide is inexpensive and can be 5’...Nature biotechnology (2016). PubMed PMID: 27136078. 2. Burgess, Shawn., et al. "Questions about NgAgo." ...stage with much-lauded CRISPR-Cas9 is DNA-guided endonuclease NgAgo! We'll discuss how NgAgo is faring with...mammalian cells and one which diminishes the endonuclease activity of the enzyme. In vivo, only nascent...someone came up with a mutant NgAgo (or a novel endonuclease) protein that can form a complex with a 5’-P-ssDNA...

...form or as ready-to-use lentivirus. Figure 2: dCas9-Tet1 demethylates cytosines at promoters and...pX330a dCas9-LSD1 for targeted removal of H3K4me1/2 and H3K9me2 histone modifications. Like the TALE-LSD1...approach for targeted DNA demethylation. Cell Discovery 2: . https://doi.org/10.1038/celldisc.2016.9 Find plasmids... Targeted DNA demethylation and activation of endogenous genes using programmable TALE-TET1 fusion proteins...-specific editing of histone modifications at endogenous enhancers. Nature Biotechnology 31:1133–1136 ...

...base pair to another base pair at a specific site. 2. Temporal, spatial, or conditional control of CRISPR...difficult-to-engineer Sulfolobus islandicus rod-shaped virus 2 (Mayo-Muñoz et al., 2018). Only viral particles that...Editing of Sulfolobus islandicus Rod-Shaped Virus 2. Viruses 10:695 . https://doi.org/10.3390/v10120695...DNA cleavage Example: AcrIIC1 binds to the HNH endonuclease domain of Cas9 and prevents target DNA cleavage...

...the result in Figure 1 could look like this: Figure 2: Expression of Plasmid B (as internal control) This...down expression of Gene X but, as shown in Figure 2, this was likely due to the original transfection ...PMID: 20074371. PubMed Central PMCID: PMC2817684. 2. Replicates and repeats—what is the difference and...produce a similar product, but do not target an endogenous gene in your experimental cells. For example,...

...Transgenesis." PLoS ONE. 8(12): e83043 (20 December 2013). 2. Carola Engler et al. "A One Pot, One Step, Precision...GreenGate are type IIS restriction endonucleases. They are like other endonucleases except that they cut at arbitrary...be very careful with the type IIS restriction endonuclease of choice, BsaI, which can be very sensitive... They finally traced the problem back to the endonuclease. “It’s just a sensitive protein,” he says. “...

... a western, proteins are: (1) separated by size, (2) transferred to a membrane, and  (3) detected using...expensive system-specific sandwiches.   Figure 2: The western blot dry apparatus includes a stack of... to the controls listed on the vendor’s website. Typically, the vendor will demonstrate that their antibody...efficiently binds to endogenously expressed protein at physiologically relevant levels. Some vendors will also test...targeting your protein of interest from a variety of vendors and you may even find plasmids encoding antibodies...attention to the validated applications listed on the vendor’s website and choose an antibody that has been validated...antibody binds to protein of the correct size. Many vendors will show that the antibody binds to transiently...