...is unique compared to the rest of the genome and (2) the sequence is located just upstream of a Protospacer...pyogenes Cas9 variants with varying PAM sequences, 2) the use of Cas9 homologs derived from species other...Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell 163:759–771 . https://doi....applications. The first non-Cas9 CRISPR protein adapted for genome engineering was Cas12a (formerly Cpf1...targeting flexibility. Cas13a (formerly C2c2) has been adapted for targeted RNA cleavage in mammalian cells. The...Replacement) system fuses Cas13b to RNA deaminase ADAR2 to create a specific RNA editor. Cas13 enzymes are...

...the absence of genome editing (Fig. 2 and 3) (5, 3). Figure 2: Gene activation with nuclease-competent...shown to exhibit more limited activity than SpCas9 (2). In many cases this is due both to lower relative...PMID: 24076762. PubMed Central PMCID: PMC3844869. 2. Chari, Raj, et al. "Unraveling CRISPR-Cas9 genome...interacts with a ≤15nt gRNA that contains an RNA adapter capable of recruiting a transcriptional activator...

... were depositing and requesting each year (Figure 2). We did a similar analysis with CRISPR pooled libraries...different categories (Figure 2C). Note that in Figure 2, the numbers on the X axis refer to distribution and...rather than a cumulative total.    Figure 2: Addgene CRISPR deposit and distribution by function...rapid adoption of new tools, illustrated in Figure 2, and the longevity of tools seen in Figure 3, indicates...our requestors. However, we can again see quick adaptation and steady use of all types of libraries, once...

... Figure 2: Ironing boards. Good as a WFH desk, and as a video control center.   Cycle 2: Zooming ...ironing board, giving it the perfect height (Fig. 2). We recorded video using a simple, external video... Figure 1: The Design-Build-Test-Learn cycle adapted for video making.   Cycle One: The Ironing ...on-camera presentations and interviews. But how can I adapt this setup for the lab, so we can get back to producing...shoot. I also added a teleprompter to the mix, adapting the setup to run some remote interviews, so the...

...handled at BSL-1, while lentivirus requires a BSL-2 setup. This requires a less involved setup when working...infection rates in your experiment. Figure 2: Visual representation of common AAV serotypes used..., 10(1). https://doi.org/10.1038/s41467-019-12449-2 Pupo, A., Fernández, A., Low, S. H., François, A.,...References and Resources References Braun, C. J., Adames, A. C., Saur, D., & Rad, R. (2022). Tutorial: design...Teyssier, N., Pan, A., Danner-Bocks, S., Sanghal, T., Yadanar, L., Tian, R., Ma, K., Palop, J. J., & Kampmann...

...therapy had fallen - plummeted really -  from grace 2 years prior with the death of Jesse Gelsinger in an... the whole field to a grinding stop and only now, 2 decades later, is it finally back in the limelight...the other hand, have broad tropism in the eye. AAV-2, the first identified serotype and the one used in...reading 1. FDA approves gene therapy for blindness 2. Gene Therapy emerges from disgrace to be the next...efficiently transduce various types of retinal neurons. Nowadays a toolbox of naturally occurring and engineered...

... to DFHBI in the presence of that metabolite (Fig.2). Spinach eventually evolved into Spinach2, a more...PMID: 25713381. PubMed Central PMCID: PMC4364232. 2. Baker, Monya. “RNA imaging in situ”. Nature Methods...emerging environmental pollutants.” Front Chem. 2014; 2: 41. PubMed PMID: 25019067. PubMed Central PMCID: ...biosensors.” TrAC Trends in Analytical Chemistry 27(2):108-117 · February 2008. 11. Svensen, Nina and Jaffrey...nucleotide-based ligand binders would prove to be highly adaptable tools. The key to aptamers’ flexibility is their...

...SMASh-tagged protein and 2) asunaprevir (small molecule). dTAG The degradation tag (dTAG) system functions...gone through an additional round of evolution (AID-2) to reduce some of the leakiness associated with the...leading to ubiquitination and proteasomal degradation. Adapted from Nabet et al., Chemical Biology, 2018...of a protein and can inducibly facilitate the degradation of the tagged protein. Degrons are destabilizing...various enzymes and sent to the proteasome for degradation. The tags we will discuss here are all conditional...conditional, meaning that they only facilitate degradation of their fused protein under certain conditions...are reversible mechanisms of control. Protein degradation can be induced by the addition of a small molecule...

...NGG-3′ (“N” can be any nucleotide base, followed by 2 guanines). This limits the use of ABEs as DNA sites... in the promoter region of gamma hemoglobin (HGB1/2) leads to continuous expression of fetal hemoglobin...evolved TadA domain. Because ABE7.10 contains a heterodimeric fusion of wild-type TadA and TadA7.10, they...wild-type TadA and the evolved TadA region. The ABE8.x-m version contains only the evolved TadA and is ~...3 components:  a deaminase (TadA, originally from E.coli, named TadA7.10 in ABE7.10)  a catalytically...nickase)  a guide RNA that targets the complex of TadA and dCas to the genomic DNA of interest  ...When ABEs are in action they bind DNA so that the TadA domain can edit the short single-stranded DNA stretch...

...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...cell differentiation (a cell type important for adaptive immune responses, which flies do not have!) (4...

...plasmid-based expression requires serial transfection of 1 or 2 plasmids that express the reprogramming factors of...for generating iPSCs by plasmid transfection where 2 of 11 clones tested had plasmid integration. Another...understanding the mechanisms of reprogramming, and 2) studies with clinical endpoints. In the first scenario...PubMed PMID: 27764162. PubMed Central PMCID: 5072705. 2. Sommer, Cesar A., and Gustavo Mostoslavsky. "The ...challenges." Journal of Cellular Physiology 228, no. 2 (2012): 267-75. PubMed PMID: 22767332.  3. Kim, Dohoon...PubMed PMID: 23546745. PubMed Central PMCID: 4176696. 2. Yusa, Kosuke, Roland Rad, Junji Takeda, and Allan...generation. Expert opinion on biological therapy, 10 2, 231-42. PubMed PMID: 20088717. PubMed Central PMCID...

...the light-inducible heterodimerizing cryptochrome 2 (CRY2) and calcium and integrin-binding protein 1 ...inactive, dead Cas9 protein (dCas9) fused to CIB1; and 2) the activator - the CRY2 photolyase homology region... fused to both the N- and C-termini of dCas9; and 2) CRY2FL-VP64, a fusion of full-length CRY2 and the...researchers saw an 11-fold increase in mRNA levels after 2 hr and a 400-fold increase after 30 hr. The system...authors fragmented Cas9 into N-terminal (residues 2-713, N713) and C-terminal (residues 714-1368, C714...nMagC714 instead of nMagHigh1-C714, generating paCas9-2. This change did not significantly alter the system...prior work, the Sato lab also showed that the paCas9-2 system could be spatially controlled and reversibly...

... working with is correct. It's also best to use 1-2 ug of the vector for digestion. PCR Design primers...plasmid; a starting point could be a molar ratio of 1:2 (plasmid:insert), but this parameter has to be optimized...colonies (5-10) and grow them in a small culture volume (2 or 3 ml) containing the corresponding antibiotic, ...provided by the major companies selling PCR reagents. Nowadays commercial polymerases are very efficient, but...

...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 ...studied in 40 labs as a great model for plant adaptation and speciation. Their phenotypes are diverse ...captivity. Sticklebacks demonstrate unusual parallel adaptation to disparate environments so can be used study...

...improve solubility and stability (Figure 2).      Figure 2: Crystal structures of CRBNmidi alone...fluorescent tags in Caenorhabditis elegans. Genetics, 228(2), iyae126. https://doi.org/10.1093/genetics/iyae126...ubiquitination substrate Ikaros (IKZF1ZF2) (B). Image adapted from Kroupova et al., 2024 under CC BY license....design and studies of protein ubiquitination and degradation.   Find the CRBNmidi plasmid here!   Kroupova...

...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...Widrick, J. J., Messemer, K. A., Troiano, E. C., Moghadaszadeh, B., Peacker, B. L., Leacock, K. A., Horwitz...

...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.... a table listing some of critical differences (adapted from Zhou and Melton, 2008, [43]): Characteristic...

...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...J. M., Xu, A., Sisley, T. A., Weissman, J. S., Adamson, B., & Liu, D. R. (2021). Efficient C•G-to-G•C ...

...and in the presence of oxidizing agents. Adapted from Fig. 2 Campbell et al, Nature Methods   Find...is always best to have options.      Fig. 2: Addgene’s western blot for mEGFP-HRas (from Plasmid...nuclear (MALAT1) and cytoplasmic (CKB1) RNA target. Adapted from Figure 3 Colognori et al 2022.   Find CRISPR...

...11 and Cas13 are 1). non-specific RNA cleavage and 2). cell toxicity. Cas7-11 does not display collateral...Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell, 163(3): 759-771 (2015). 10.1016...features and evolved PAM sites, making Cas9 an adaptable choice for many genome engineering endeavors. ...target sequence and initiates single stranded DNA degradation. Cas3 DNA digestion can proceed up to multiple...   Choosing a Cas While Cas9 may be the most adaptable Cas currently available, the other Cas proteins...