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  1. The PAM Requirement and Expanding CRISPR Beyond SpCas9

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    Blog Post
    ...Addgene. Kim D, Kim J, Hur JK, Been KW, Yoon S, Kim J-S (2016) Genome-wide analysis reveals specificities...Cho H-Y, Song DW, Lee KJ, Jung MH, Kim S, Kim JH, Kim JH, Kim J-S (2017) In vivo genome editing with a ...sequences While PAM sequences for the commonly used S. pyogenes Cas9 (3'-NGG) are abundant throughout the...to circumvent this limitation: 1) the use of novel S. pyogenes Cas9 variants with varying PAM sequences... of Cas9 homologs derived from species other than S. pyogenes, and 3) the use of non-Cas9 enzymes. (For...site, check out this video from IGI!) Synthetic S. pyogenes Cas9s with novel PAM recognition In 2015...selection screens in bacteria to identify mutants of S. pyogenes Cas9 that were able to cleave target DNA...

  2. Viral Vectors 101: AAV Serotypes and Tissue Tropism

    Type
    Blog Post
    ...B.-Y., Viswanathan, S., Gaj, T., Lavzin, M., Ritola, K. D., Lindo, S., Michael, S., Kuleshova, E., Ojala...resources References Ahuja, V., Jeyabalan, S., & Tzanakakis, E. S. (2025). Evaluation of transduction efficiency...https://doi.org/10.1038/s41467-023-39554-7 Issa, S. S., Shaimardanova, A. A., Solovyeva, V. V., & Rizvanov... L., Marhic, A., Chapart, M., Vasseur, S., Benkhelifa-Ziyyat, S., Bigot, A., Butler-Browne, G., Mouly,...doi.org/10.1089/hum.2019.173 Pillay, S., Meyer, N. L., Puschnik, A. S., Davulcu, O., Diep, J., Ishikawa,...j.neuron.2016.09.021 Walters, R. W., Yi, S. M., Keshavjee, S., Brown, K. E., Welsh, M. J., Chiorini, J...., Wong, A. C. H., Gokal, D., Feng, Y., Tabar, M. S., Metierre, C., Parsania, C., Song, X., Wang, G., ...

  3. Viral Vectors 101: AAV Variables That Matter

    Type
    Blog Post
    ...and happy optimizing!  Recommended Reading Issa, S. S., Shaimardanova, A. A., Solovyeva, V. V., & Rizvanov...Resources References Aschauer, D. F., Kreuz, S., & Rumpel, S. (2013). Analysis of Transduction Efficiency...10.1089/hum.2009.169 Dudek, A. M., Pillay, S., Puschnik, A. S., Nagamine, C. M., Cheng, F., Qiu, J., Carette... https://doi.org/10.1038/s41434-019-0075-6 Issa, S. S., Shaimardanova, A. A., Solovyeva, V. V., & Rizvanov...10.1371/journal.pone.0076310 Damdindorj, L., Karnan, S., Ota, A., Hossain, E., Konishi, Y., Hosokawa, Y.,...Fong, D. M., Mouravlev, A., Young, D., & O’Carroll, S. J. (2019). Astrocyte-selective AAV gene therapy through...cells12050785 Kanaan, N. M., Sellnow, R. C., Boye, S. L., Coberly, B., Bennett, A., Agbandje-McKenna, M...

  4. Cpf1: A New Tool for CRISPR Genome Editing

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    Blog Post
    ...depletion assay to discover FnCpf1’s PAM sequence requirements. Cpf1’s preferred PAM is 5’-TTN, differing...RuvC-like endonuclease domain, but they lack Cas9’s second HNH endonuclease domain. Cpf1 cleaves DNA in... RuvC-like endonuclease domain, but it lacks Cas9’s other HNH endonuclease domain, indicating that Cpf1... nuclease could potentially overcome some of Cas9’s shortcomings - namely its blunt double stranded cleavage...nucleotides in length, about the same size as Cas9’s, but with the direct repeat preceding the spacer rather...crRNA is also much simpler in structure than Cas9’s; only a short stem-loop structure in the direct repeat... added another option to the CRISPR toolbox. Cpf1’s staggered cleavage pattern opens up the possibility...

  5. The Pleiades Promoter Project

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    ...Jiang, S., Khorasan-zadeh, S., Komljenovic, I., Laprise, S., Liao, N. Y., Lim, J. S., Lithwick, S., Liu..., Wong, B. K., Wong, S. H., Wong, T. Y., Yang, G. S., Ypsilanti, A. R., Jones, S. J., Holt, R. A., Goldowitz... Ho Sui, S. J. Fulton, D. L., Ali, J., Amirabbasi, M., Arenillas, D. J., Babyak, N., Black, S. F., Bonaguro...selective expression in the brain. Proc Natl Acad Sci U S A, 107 (38):16589-94. https://doi.org/10.1073/pnas...

  6. Tag Your Favorite Yeast Genes with Ease

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    Blog Post
    ...integrate protein tags into the genomes of S. cerevisiae and S. pombe. The protocol is surprisingly simple...Yeast-optimized fluorophores for imaging Lee S, et al. (2013) PLoS ONE 8(7): e67902. Many imaging...al.(1) assessed many of these fluorescent tags in S. cerevisiae, looking at their performance in categories...plasmids for a wide variety of genome modifications in S. Pombe, including full and partial gene deletion, ...describe a complimentary set of plasmids for use in S. cerevisiae, with the additional benefit of multiple...system that isn't mentioned here? References: Lee S, Lim WA, Thorn KS. PLoS One. 2013 Jul 2;8(7):e67902...

  7. CRISPR References and Information

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    ...identifies putative target sites for S. pyogenes Cas9, S. aureus Cas9, S. thermophilus Cas9, N. meningitidis...checks for off-target binding and can work for S. pyogenes , S. thermophilus , or N. meningitidis Cas9 PAMs...support for bacteria ( E. coli, B. subtilis ), yeast ( S. cerevisiae ), worm ( C. elegans ), fruit fly, zebrafish...to maximize on-target activity for selected target(s). Uses human, mouse, and rat reference genomes. Hosted...forum started by the Feng Zhang Lab . Protocols Lab(s) Description Plasmids in protocol Download protocol...gRNA: pSimpleII-U6-tracr-U6-BsmBI-NLS-NmCas9-HA-NLS(s) PDF, 49 KB Vosshall and Matthews (Link opens in a...

  8. p53 Pathway

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    ...ageing-associated phenotypes. Tyner SD, Venkatachalam S, Choi J, Jones S, Ghebranious N, Igelmann H, Lu X, Soron G...papillomavirus). These mutations interfere with p53’s ability to activate transcription, and they also have...through oligomerization. In particular, the loss of p53’s pro-apoptotic effects is especially important to tumorigenesis...kinase ATR ATR serine/threonine kinase B99 G-2 and S-phase expressed 1; also known as GTSE1 BAI-1 Brain-specific... human malignancy: a clinical perspective. Surget S, Khoury MP, Bourdon JC. Onco Targets Ther. 2013 Dec...

  9. Live and Let Dye: Self-Labeling Protein Tags

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    ...Erdmann, R. S., Baguley, S. W., Richens, J. H., Wissner, R. F., Xi, Z., Allgeyer, E. S., Zhong, S., Thompson...F., Kromann, E. B., Allgeyer, E. S., Erdmann, R. S., Wood Baguley, S., Sirinakis, G., Schepartz, A., Baddeley...doi.org/10.1016/j.chembiol.2019.01.003 Gallagher, S. S., Sable, J. E., Sheetz, M. P., & Cornish, V. W. ... R., Bewersdorf, J., Rothman, J. E., Johnston, D. S., Schepartz, A., & Toomre, D. (2019). Labeling Strategies...10.1016/j.chembiol.2008.01.007 Griffin, B. A., Adams, S. R., & Tsien, R. Y. (1998). Specific Covalent Labeling...doi.org/10.1021/ar200099f Keppler, A., Gendreizig, S., Gronemeyer, T., Pick, H., Vogel, H., & Johnsson,...s41467-023-40788-8 Ulrichs, H., Gaska, I., & Shekhar, S. (2023). Multicomponent regulation of actin barbed...

  10. CRISPR History and Development for Genome Engineering

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    ...(monocots and dicots) C. elegans Yeast ( S. cerevisiae and S. pombe ) Zebrafish Xenopus References Barrangou... Acad Sci U S A . 112(10):3002-7. PMID: 25713381 Ma H, Tu LC, Naseri A, Huisman M, Zhang S, Grunwald D...and number/types of Cas proteins. Makarova et al. ’s classification defines 5 types and 16 subtypes based...PAM, improving targeting in AT-rich genomes. Cpf1's small size also makes it suitable for multiplexing...Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P. 2007. CRISPR provides acquired...12. PMID: 17379808 Bondy-Denomy J, Garcia B, Strum S, Du M, Rollins MF, Hidalgo-Reyes Y, Wiedenheft B, ...407-410. PMID: 28931002 Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini...

  11. Zhang Lab CRISPR Page

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    ... CY, Gootenberg JS, Konermann S, Trevino AE, Scott DA, Inoue A, Matoba S, Zhang Y, Zhang F. Cell . 2013...Mouse CRISPR ( C lustered R egularly I nterspaced S hort P alindromic R epeats) is a microbial nuclease...implemented in mammalian cells by co-expressing the S. pyogenes Cas9 (SpCas9) nuclease along with the guide...The single vector system uses a smaller Cas9 from S. aureus (SaCas9) that has been human codon-optimized...et al., Nature 2015). The dual vector system uses S. pyogenes Cas9 (SpCas9), using one vector to express...using CRISPR/Cas Systems. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini...Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, Cradick...

  12. Of Myc and Men

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    Blog Post
    ...Schüchner, et al.’s results mean the end of 9E10’s time in the saga of the Myc tag? Hardly! 9E10’s accessibility...generated in an attempt to overcome some of Myc1-9E10’s shortcomings. Wait, 9E10 isn’t the Mary Poppins of... acids neighboring the Myc epitope influence 9E10’s affinity for the epitope, a phenomenon known as sequence...https://doi.org/10.1128/mcb.5.12.3610-3616.1985 Munro S, Pelham HR (1984) Use of peptide tagging to detect...doi.org/10.1002/j.1460-2075.1984.tb02263.x  Munro S, Pelham HRB (1986) An hsp70-like protein in the ER...https://doi.org/10.1016/0092-8674(86)90746-4 Munro S, Pelham HRB (1987) A C-terminal signal prevents secretion...//doi.org/10.1016/0092-8674(87)90086-9  Schüchner S, Behm C, Mudrak I, Ogris E (2020) The Myc tag monoclonal...

  13. Viral Vectors 101: Systemic Capsids

    Type
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    ... Kumar, S., Chen, X., Goertsen, D., Coughlin, G. M., Hori, A. M., Chuapoco, M. R., Otis, T. S., Miles,...Ichiki, T., Mazmanian, S. K., Osborne, P. B., Keast, J. R., Miller, C. T., Fox, A. S., Chiu, I. M., & Gradinaru... Bindu, D. S., Zhang, M., Taskin, N., Goertsen, D., Shay, T. F., Sullivan, E. E., Huang, S.-F., Ravindra...B. P., Kumar, S. R., Chan, K. Y., Banerjee, A., Wu, W.-L., Yang, B., Huber, N., Pasca, S. P., & Gradinaru...., Ng, C., Nammour, J., Maiz, D., Dujardin, S., Ramirez, S. H., Hudry, E., & Maguire, C. A. (2019). Selection...Liguore, W. A., Domire, J. S., Button, D., Wang, Y., Dufour, B. D., Srinivasan, S., & McBride, J. L. (2019...Stanton, A., King, E. M., Ye, S., Tellez, L., Krunnfusz, A., Tavakoli, S., Widrick, J. J., Messemer, K...

  14. CRISPR 101: Cytosine and Adenine Base Editors

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    ...Zhang, S., Wu, H., Gao, H., Bai, S., Li, C., Ding, R., Xue, N., Lei, Z., Chen, Y., Guan, Y., Siwko, S., …...Edwards, A., Gehrke, J. M., Lee, S.-J., Liquori, A. J., Murray, R., Packer, M. S., Rinaldi, C., Slaymaker, I...Grünewald, J., Zhou, R., Lareau, C. A., Garcia, S. P., Iyer, S., Miller, B. R., Langner, L. M., Hsu, J. Y....McElroy, A. N., Pandey, S., Doman, J. L., Huang, T. P., Raguram, A., Banskota, S., Newby, G. A., Tolar,...Gaudelli, N. M., Komor, A. C., Rees, H. A., Packer, M. S., Badran, A. H., Bryson, D. I., & Liu, D. R. (2017...Kim, Y. B., Komor, A. C., Levy, J. M., Packer, M. S., Zhao, K. T., & Liu, D. R. (2017). Increasing the...10.1038/nbt.4172 Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A., & Liu, D. R. (2016). Programmable ...

  15. CRISPR 101: Making a Knock-In Cell Line

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    ...viability of your cells. Enrich S-phase cells HR operates predominantly in S and G2 phase of the cell cycle....1016/j.jbiotec.2019.11.010 Wright, D. W., Shah, S. S., Heyer, W. D. Homologous recombination and the ...2018). 1 10.1074/jbc.TM118.000372 Yang, H., Ren, S., Yu, S., Pan, H., et al. Methods favoring homology-directed...regulatory proteins responsible for the transition out of S phase, so cells stay in an HDR-promoted cycle longer...., 21(18): 6461 (2020). 10.3390/ijms21186461 Lin, S., Staahl, B. T., Alla, R. K., Doudna, J. A. Enhanced...01836-2 Charpentier, M., Khedher, A. H. Y., Menerot, S., Brion, A., et al. CtIP fusion to Cas9 enhances transgene...

  16. Viral Vectors 101: Optogenetic Tools

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    ...10.1038/nn.2495 Marshel, J. H., Kim, Y. S., Machado, T. A., Quirin, S., Benson, B., Kadmon, J., Raja, C., ..., J. C., McKnight, D. J., Yoshizawa, S., Kato, H. E., Ganguli, S., & Deisseroth, K. (2019). Cortical layer–specific...following factors will help you determine which one(s) are candidates for your experiments.  Wavelengths....1007/978-1-0716-0755-8_2 Lehtinen, K., Nokia, M. S., & Takala, H. (2022). Red Light Optogenetics in Neuroscience...://doi.org/10.1007/978-1-0716-0755-8_2 Boyden, E. S., Zhang, F., Bamberg, E., Nagel, G., & Deisseroth,...Gunaydin, L. A., Yizhar, O., Berndt, A., Sohal, V. S., Deisseroth, K., & Hegemann, P. (2010). Ultrafast....1808 Nagel, G., Szellas, T., Huhn, W., Kateriya, S., Adeishvili, N., Berthold, P., Ollig, D., Hegemann...

  17. The AAVantages of AAV in CRISPR Screens

    Type
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    ... Hanlon, K. S., Kleinstiver, B. P., Garcia, S. P., Zaborowski, M. P., Volak, A., Spirig, S. E., Muller...Sousa, A. A., Tsai, S. Q., Bengtsson, N. E., Lööv, C., Ingelsson, M., Chamberlain, J. S., Corey, D. P., Aryee.../s41467-019-12449-2 Pupo, A., Fernández, A., Low, S. H., François, A., Suárez-Amarán, L., & Samulski, ...Rose, I. V., Teyssier, N., Pan, A., Danner-Bocks, S., Sanghal, T., Yadanar, L., Tian, R., Ma, K., Palop... J., Klingler, E., Kuhn, M., Farouni, R., Lagler, S., Kalamakis, G., Lischetti, U., Jabaudon, D., & Platt...

  18. Rinehart Lab Phosphoprotein Reagents

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    ...Aguiar, M., Beausoleil, S. A., Paulo, J. A., Rinehart, J., Huttlin, E. L., & Gygi, S. P. (2022). A multi-... described in: Mohler, K., Moen, J. M., Rogulina, S., & Rinehart, J. (2023). System‐wide optimization ...Barber, K. W., Muir, P., Szeligowski, R. V., Rogulina, S., Gerstein, M., Sampson, J. R., Isaacs, F. J., & Rinehart...Aerni, H. R., J, N., MA, Haimovich, A. D., Rogulina, S., Isaacs, F. J., & Rinehart, J. (2015). A flexible... N. L., Barber, K. W., Ter Haar, C. M., Rogulina, S., Amrofell, M. B., Isaacs, F. J., Rinehart, J., & ...Heinemann, I. U., Rovner, A. J., Aerni, H. R., Rogulina, S., Cheng, L., Olds, W., Fischer, J. T., Söll, D., Isaacs...

  19. X-CHIME: Context Dependent Germline Knockout in Immune Cells

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    ...-excited GFP (vex) Lineage-Cre; Rosa26-Cas9 S-CHIME Knocks out pairs of genes sequentially pXPR...hindering studies post embryogenesis. Using C-CHIME and S-CHIME, we discovered that double knockout of PTPN1...References LaFleur, M. W., Lemmen, A. M., Streeter, I. S. L., Nguyen, T. H., Milling, L. E., Derosia, N. M...., Z. M., Gillis, J. E., Tjokrosurjo, Q., Markson, S. C., Huang, A. Y., Anekal, P. V., Montero Llopis, ...Coxe, M. A., Yates, K. B., Trombley, J. D., Weiss, S. A., Brown, F. D., Gillis, J. E., Coxe, D. J., Doench...

  20. Deisseroth INTRSECT Collection

    Type
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    ...) Chuhma N, Mingote S, Yetnikoff L, Kalmbach A, Ma T, Ztaou S, Sienna AC, Tepler S, Poulin JF, Ansorge...Link opens in a new window) Mingote S, Amsellem A, Kempf A, Rayport S, Chuhma N. 2020. Dopamine-glutamate...Ansorge M, Awatramani R, Kang UJ, Rayport S. 2018. Dopamine neuron glutamate cotransmission evokes a delayed...Preprint (Link opens in a new window) Yu K, Ahrens S, Zhang X, Schiff H, Ramakrishnan C, Fenno L, Deisseroth...Ramakrishnan C, Fenno L, Deisseroth K, Herry C, Arber S, Lüthi A. 2016. Midbrain circuits for defensive behaviour...
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