...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...

...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...

... 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...

...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...

...native enzyme, which are summarized in Table 2 below.  Table 2: Properties of native luciferases Organism...ostracod(Cypridina noctiluca)Sea firefly(Cypridina hilgendorfii) 465 62 Cypridina (Vargulin) luciferin None ...

...host range plasmids. FEMS Microbiology Letters, 348(2), 87–96. https://doi.org/10.1111/1574-6968.12241  ...mobilization of broad host-range IncQ plasmids. Plasmid, 62(2), 57–70. https://doi.org/10.1016/j.plasmid.2009.05.001...negative hosts. Biotechnology and Bioengineering, 106(2), 326–332. https://doi.org/10.1002/bit.22695  Schmidhauser...Physical and genetic studies with restriction endonucleases on the broad host-range plasmid RK2. Molecular...

...presented in a series of quick (10 minute) or very quick (2 minute) talks so we got to hear from everyone. Addgene...evolutionary tree at least 6-7 times and on at least 2 separate continents. Mimulus (Andrea Sweigart) Monkeyflowers...Organisms, Part 1 Five Popular Model Organisms, Part 2 ...lab has the capability) with receptor mediated endocytosis. They used this to edited those brown marmorated...

...to a specific area at the plasma membrane (Figure 2). Just express the plasma membrane-localized iLID ...cells will be ready for the spotlight.    Figure 2: Opto-RhoGEFs to control Rho GTPase activity. A) Schematic...OptoTIAM (magenta) expressed in a monolayer of endothelial cells, showing cell protrusions and overlaps ...junction integrity in the vascular barrier of the endothelium, but they could be applied to study other actin-dependent...provided CIB constructs for targeting Golgi, ER, and endosome membranes) (Figure 4). The authors used SuperPLD...scale, enabling precise control over vascular endothelial barrier strength. eLife, 12, RP84364. https:/...

...expressed as a fusion with your favorite protein and (2) a synthetic fluorophore-contianing “ligand” that ...cell 58.4 (2015): 644-659. PubMed PMID: 26000849. 2. Grimm, Jonathan B., et al. "A general method to improve...conjugated to molecular species with high affinity for endogenous molecular targets such as paclitaxel. Unlike ...required as the small-molecule binding motif targets endogenous protein (Figure 1f). Labeling strategy pros...

...green squares, purple circles and blue triangles. 2) An antibody immobilized on a bead binds specifically...protein G, a novel IgG-binding reagent. J Immunol 133(2):969-974. Hjelm H, Hjelm K, Sjöquist J (1972) Protein...typically expressed at much higher levels than endogenous proteins. Data obtained from experiments using...tagged protein may not be translatable to the endogenous system and will need to be verified using alternative...

...is always best to have options.      Fig. 2: Addgene’s western blot for mEGFP-HRas (from Plasmid...the presence of oxidizing agents. Adapted from Fig. 2 Campbell et al, Nature Methods   Find YFP plasmids...also demonstrated the ability to be used as an endogenous RNA imaging tool.   Fig. 1: Csm, shRNA...

... as muscular dystrophies, heart disease, and type 2 diabetes, across four different types of human cells...(#186715) and hCtRNA_VT (#186716).   Fig. 2: Comparison of previous representative dual-gRNA architectures...expression, the individual gRNAs are released by endogenous tRNA processing machineries, specifically, RNaseZ...

...while increasing the standard mutation rate by only ~2-fold over nCas9 levels. Two sets of modifications ...10-3 mutations per nucleotide per generation, and (2) the addition of the thioredoxin-binding domain (TBD...extends it from the 3’ end, while its native endonuclease activity degrades the displaced strand.   ...