...possible when each of the XFP genes is introduced at ~2 copies per cell per color, following a Poisson distribution...(prepared with Addgene #104110 - #104112) (Figure 2). We’ve also created constructs with containing chemical...AAV titer - The XFP genes have to be introduced at ~2 copies per cell per color to achieve the highest color...electroporation. Then at your preferred time points, sacrifice the animals and fix the samples. When you use ...AAV titer for the best results. The timing of sacrifice when using Tetbow AAVs - You will have to wait...

...or non-homologous end joining (see Figure 2).  Figure 2: Schematic representation of integration...due to insertional mutagenesis (Howe et al., 2008). 2. Transient expression of transgenes/shRNAs If you ...vectors: progress and applications. Gene Therapy, 17(2), 150–157. https://doi.org/10.1038/gt.2009.135. PubMed...efficiently during infection. Journal of Virology, 70(2), 721–728. https://doi.org/10.1128/JVI.70.2.721-728.1996...molecular biology for over three decades. Widely used across various research applications, they have become...lentiviral vectors, the lentiviral genome is distributed across multiple plasmids. For simplicity, we’ll use the...

...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...refresher if you ever lose track of which acronym is which. Acronym Full name Notes CRISPR terms gRNA... of CBEs and ABEs. This post includes a lot of acronyms, so we’ve included a glossary at the end if you...

... used. Follow the manufacturer’s instructions.   2. Isolate Your Insert and Vector by Gel Purification...DNA concentration alone. One method is to conduct 2 ligations for each plasmid you are trying to create...containing the ligation mixture with backbone alone; 2. Transformation containing ligation mixture with insert...colonies than the vector + insert plate (unsuccessful 2), or roughly equivalent numbers of colonies on each... or equivalent to backbone + insert, unsuccessful 2 and 3 above), you can try ligating the recipient plasmid... gel purification, you use a voltage difference across a gel matrix (usually agarose) to pull your negatively...cathode (- charge). When the voltage is applied across the gel, the DNA migrates toward the anode (+ charge...

...systems: a burst of class 2 and derived variants. Nature Reviews Microbiology, 18(2), 67–83. https://doi.org...resource that provides summary information on Class 2 Cas enzymes. The information is organized in wiki ...this content and much, much more.     Fig. 2 – List of resources and properties for a Cas entry...Database: A Functional Classification System for Class 2 CRISPR-Cas Enzymes [Preprint]. Biochemistry, Biophysics...required? CasPEDIA provides information on crRNA and tracrRNA requirements, along with any other required non-CRISPR-associated...

...bottom of the image. Stem loops 1 and 2 extend horizontally across both lobes. The guide RNA in this structure...Cas9 from Streptococcus pyogenes.   Figure 2:  Crystal structure of S. pyogenes Cas9 in the apo...NUC and REC lobes laying open like a book (Figure 2). The portion of the CTD that recognizes the PAM is...it doesn’t appear in the crystal structure (Figure 2) at all. When this crucial recognition region is disordered...regions, so crystal structures like those in Figures 2 and 3 don’t show these regions, even if they are present...is required for Cas9 nuclease function. Stem loops 2 and 3 are not strictly necessary for in vitro cleavage...open conformation, but highly flexible, as in Figure 2. The guide RNA is bound primarily by the REC lobe,...

...Originally published Dec 7, 2017 and updated Jul 2, 2020. Promoters may be the star of gene regulation...sequence targeted in the experiment. Figure 2: Comparing CAPTURE and CAPTURE 2.0.     Original...cell line expressing 1) FLAG-biotin-tagged dCas9, 2) BirA, and 3) one or more gRNAs Two separate lentiviruses...delivering 1) a BioTAP-tagged dCas9 and an eGFP tag, and 2) gRNAs Number of loci targeted One Many Sample...identified and the level of on-target enrichment. 2. Determining the spatial and hierarchical organization...Cytogenetics, 11. https://doi.org/10.1186/s13039-018-0368-2  Liu X, Zhang Y, Chen Y, Li M, Zhou F, Li K, Cao H...cumbersome and also makes it hard to compare results across multiple targets because different CAPTURE cell...

...for the 2+ million DHS sites. See below for further details. DNA is a 3D Fractal Figure 2: DNA compaction...: 1. Find Enhancers that have NANOG binding sites 2. Design a CRISPR screen to target and disrupt the ...NANOG enhancers 3. CRISPR out the enhancers from step 2. See which ESCs die or differentiate Step 3 reveals...expression for NANOG enhancers, we’re ready for step 2: design a CRISPR screen to disrupt these enhancers...to CRISPR out the enhancers we identified in step 2. The result is a heterogenous population of stem cells...measure the relative proportion of guide RNAs (gRNAs) across the population. Recall that guide RNAs are the ...

...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...

... H. J. (2009). Rats! Disease Models & Mechanisms, 2(5–6), 206–210. https://doi.org/10.1242/dmm.002733 ...regulate regeneration and wound healing are conserved across species, studying the axolotl may reveal the potential...

...low expression, and everything in between (Figure 2). These smears tend to occur when you are looking ...based on your experiment. You can see from Figure 2 that determining where to place your gate based on...the dot plot would be complicated.  Figure 2: (A) a smeary dot plot showing a range of antibody...plot in Figure 4 compared to the dot plot in Figure 2. Quantification using MFI makes it easier to identify...interest: two clear populations or a huge smear across your FSH vs reporter plot. In this post, I’ll walk...

... (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 ...fewer off-target cuts when compared to WT SpCas9 across a variety of genomic sites. Knowledge of Cas9’s...activity and similar or decreased off-target activity across the sites tested when compared to eSpCas9(1.1) ...

... flanking sequences to their input string (Figure 2). This string can then be used as input for the prediction...top 5) and test them in their labs. Figure 2: Guidelines to predict pegRNA efficiencies with pridict.it...Biotechnology. https://doi.org/10.1038/s41587-024-02268-2 Schep, R., Brinkman, E. K., Leemans, C., Vergara, X., ...2023), their predictive power is sometimes limited across different targets due to variations in chromatin...chromatin substantially affects prime editing outcomes across many genomic locations. In our study, we used the...learning prediction of prime editing efficiency across diverse chromatin contexts. Nature Biotechnology...

...upstream of an opsin gene (e.g., channelrhodopsin-2, ChR2) fused to a reporter gene (e.g., mCherry) to...the correct orientation to be transcribed (Figure 2) (Sharma and Zhu, 2014). How does FLEx enable strong... Abdallah, Khaled, et al. "Adeno-associated virus 2/9 delivery of Cre recombinase in mouse primary afferents...Deniz, et al. "A FLEX switch targets Channelrhodopsin-2 to multiple cell types for imaging and long-range ...These proteins control the flow of electric charges across cell membranes and maintain membrane potential ...stimulations are triggered by the movement of ions across the axon's membrane: once a certain number of positive...wavelength, microbial rhodopsins enable ions to flow across the axon’s membrane, thereby controlling neural...

...//doi.org/10.1002/1096-8628(200022)97:2<147::aid-ajmg6>3.0.co;2-o Baker SA, Chen L, Wilkins AD, Yu P, ...Caused by a mutation in the methyl-CpG binding protein 2 (MECP2) gene, Rett Syndrome is a rare, progressive...X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat Genet 23:185–188 . https://doi.org/10.1038/13810... (2000) Rett syndrome: Methyl-CpG-binding protein 2 mutations and phenotype-genotype correlations. Am ...RNA editing can repair a gene in a living animal across multiple cell types using a mouse model for a human...

...Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system." Cell (2015). PubMed PMID: 26422227... Find plasmids from this publication at Addgene. 2. Kleinstiver, Benjamin P., et al. "Genome-wide specificities...Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell. 2015 Sep 23. pii: S0092-8674...and requires only one RNA rather than the two (tracrRNA and crRNA) needed by Cas9 for cleavage. Now, two...require HDR over NHEJ, so comparing HDR efficiency across CRISPR nucleases and NgAgo is an important next...

... this problem? (2-3 sentences) What will be the focus of your first grant? (1-2 sentences) ...of scientific success is publications and grants. 2. Write succinct and easy to understand documents A...consideration. 3. Present an easy to follow seminar See item 2. Attendees will include grad students (that are solicited...people would agree is important to understand. (1-2 sentences). For example: Distinguishing correctly ...practical and accessible. If there's something you come across that is not covered, please email me and I'll try...

... Multiple Introns.” Plant Communications, vol. 2, no. 2, Elsevier BV, Mar. 2021, p. 100135. Crossref, ...Constructs.” PLoS ONE, edited by Jean Peccoud, vol. 6, no. 2, Public Library of Science (PLoS), 18 Feb. 2011, p... vol. 5, no. 10, American Chemical Society (ACS), 2 May 2016, pp. 1059–69. Crossref, https://doi.org/10.1021...of Plant Genomes.” The Plant Genome, vol. 16, no. 2, Wiley, 9 Mar. 2023. Crossref, https://doi.org/10.1002...Crossref, https://doi.org/10.1186/s12870-020-02388-2. Stuttmann, Johannes, et al. “Highly Efficient Multiplex...Crossref, https://doi.org/10.1038/s41598-019-46171-2. Resources on Addgene.org Modular Cloning (MoClo...as promoters or fluorophores are interchangeable across organisms. When using the MK system, many of the...

... the two DNA strands and releasing topoisomerase (2).  These days commercially available TOPO kits provide...Taq polymerase using your favorite PCR protocol.  2. Set Up TOPO Cloning Reaction: Mix together the PCR...PubMed PMID: 1658796. PubMed Central PMCID: PMC52876. 2. Novel approach to molecular cloning and polynucleotide... Perform Site Directed Mutagenesis by PCR Use REPLACR Mutatgenesis to Easily Generate Insertion and Deletions...

... However, if you have a larger knock-in (200 bp – 2 kb), a dsDNA donor will need to be used due to synthesis...accidentally change your protein of interest.   Fig. 2: Stop re-cutting with PAM and guide RNA disrupting... the other repair mechanisms Mammalian cells have 2 primary repair mechanisms besides HR - Non-homologous.... Com., 8(2024), (2017). 10.1038/s41467-017-01836-2 Charpentier, M., Khedher, A. H. Y., Menerot, S., Brion... than 10% of DNA repair, with this rate varying across cell types. Variance can be caused by cell cycle...