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TypeBlog PostPublishedJan. 13, 2025, 9:49 p.m.... A., Chan, M. M., Bauer, D. E., Marson, A., Parsons, L. R., & Adamson, B. (2024). Improving prime editing... Chen, C., Nelson, J. W., Newby, G. A., Sahin, M., Osborn, M. J., Weissman, J. S., Adamson, B., & Liu,.... coli Ec48 retron RT and the S. pombe Tf1 retrotransposon RT, respectively. Their small size comes at... even in contexts where expression was reduced (Nelson et al., 2022). PE7: An alternate approach to RNA...RNA stability This recent contribution by the Adamson lab also addresses the problem of RNA stability....References Anzalone, A. V., Gao, X. D., Podracky, C. J., Nelson, A. T., Koblan, L. W., Raguram, A., Levy, J. M....A. A., Koblan, L. W., Levy, J. M., Chen, P. J., Wilson, C., Newby, G. A., Raguram, A., & Liu, D. R. (2019...
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Hot Plasmids: Spring 2025
TypeBlog PostPublishedJune 3, 2025, 1:15 p.m....fused to a shortened R2Tocc transposon protein, and can "retarget" the transposon to new loci in the genome... Image courtesy of IPI. Repurposing retrotransposons for DNA editing By Emily P. Bentley For RNA-guided...beyond Cas9 with STITCHR: a combined CRISPR / retrotransposon system with the pithy name "site-specific target-primed...this tool based on several new site-specific retrotransposon elements they identified and characterized ...characterized (Fell et al., 2025). In retrotransposon insertion, a combined endonuclease-reverse transcriptase uses...genome. The modified transposon RNA has untranslated regions (U, blue) on the outside of the homology arms...(teal). The Cas9 gRNA targeting guide and the transposon RNA homology both help direct the fusion construct... -
Design Tips for Prime Editing
TypeBlog PostPublishedJan. 23, 2025, 2:15 p.m.... A., Chan, M. M., Bauer, D. E., Marson, A., Parsons, L. R., & Adamson, B. (2024). Improving prime editing... Chen, C., Nelson, J. W., Newby, G. A., Sahin, M., Osborn, M. J., Weissman, J. S., Adamson, B., & Liu,...-RNA base pairing with the primer binding site (Nelson et al., 2022). PE7: This editor includes the fused...A. A., Koblan, L. W., Levy, J. M., Chen, P. J., Wilson, C., Newby, G. A., Raguram, A., & Liu, D. R. (2019...e29. https://doi.org/10.1016/j.cell.2021.09.018 Nelson, J. W., Randolph, P. B., Shen, S. P., Everette,...Ravisankar, P., Ward, C. C., Levesque, S., Jing, Y., Simpson, D., Zhao, A., Li, H., Yan, W., Goudy, L., Schmidt... -
Live and Let Dye: Self-Labeling Protein Tags
TypeBlog PostPublishedApril 16, 2024, 1:15 p.m...., based on resorufin). SNAP-tag: In 2003, Kai Johnsson’s lab developed a self-labeling fusion tag based...Addgene’s collection. CLIP-tag: Also in 2008, Kai Johnsson’s lab reported another AGT-based tag named CLIP-tag...STED) nanoscopy. Scale bar, 2 μm. Inset shows comparison with standard confocal microscopy. Image adapted...159 aa) are smaller than HaloTag (295 aa) – for comparison, common fluorescent proteins are typically 230...Wissner, R. F., Xi, Z., Allgeyer, E. S., Zhong, S., Thompson, A. D., Lowe, N., Butler, R., Bewersdorf, J., ...Strategies Matter for Super-Resolution Microscopy: A Comparison between HaloTags and SNAP-tags. Cell Chemical...Corrêa, I. R., Kindermann, M., Beaufils, F., & Johnsson, K. (2008). An Engineered Protein Tag for Multiprotein... -
22 Hot Plasmid Technologies from 2014
TypeBlog PostPublishedJan. 6, 2015, 5:21 p.m....new variant was named ChrimsonR. Plasmids containing Chronos, Chrimson, ChrimsonR, CsChR, and CoChR have...inactivation. Find the Robinson plasmids or the Royle plasmids and get to work! Robinson et al., Dev Cell...Updated mini-transposon vector for bacterial mutagenesis or gene targeting Victor de Lorenzo's lab...selecting the important elements from existing transposon and vector systems and creating an all-synthetic...British idiom for “taking by surprise”) by Margaret Robinson’s lab, and more recently used by Stephen Royle’...Shining a light on channelrhodopsins – Chronos & Chrimson Through de novo sequencing of 127 algal transcriptomes...include a pair of channelrhodopsins, Chronos and Chrimson, which have enabled the group to perform independent... -
Transferable Skills: Negotiation
TypeBlog PostPublishedMarch 3, 2022, 2 p.m....everyone wins. What are the underlying reasons for this person’s request? Your colleague is if someone...need help from another person/team at the company. How do I negotiate with the person/team for help? What...can help you understand the perspective of all persons involved and be used as a guide to point you toward...important plans? What is most important to each person? Someone has to be the first author but another...agrees to pick up both children while the other person uses an available after-hours time slot. You were...thoughtful about what is most important to each person. It can especially be difficult to think creatively...supervisors and lead to you being remembered as a person who tries to find solutions that benefit everyone... -
CRISPR Methods for Bacteria: Genome Engineering, CRISPRa, CRISPRi, Base Editing, and More
TypeBlog PostPublishedSept. 28, 2020, noon...modify a given gene. CRISPR-tranposons By combining CRISPR editing and transposons, the Sternberg and Zhang...pneumoniae. Robert Husson lab's M. tuberculosis CRISPRi plasmids A group from the Husson lab used CRISPRi...insert extra elements, like a repair template, using Gibson Assembly or restriction enzyme cloning. Streptomyces...Zhang labs developed CRISPR-transposons. The Sternberg lab's system called INTEGRATE (Insertion of Transposable...By using a multi-spacer CRISPR array, CRISPR-transposons can be multiplexed. Transcriptional repression...CRISPRi for Gram-negative and Gram-positive bacteria Jason Peters, Oren Rosenberg, and Carol Gross used two...https://doi.org/10.1038/s41564-018-0327-z Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP... -
MXS Chaining
TypeBlog PostPublishedFeb. 7, 2017, 3:30 p.m....compatible cohesive ends Engler et al., 2008 Gibson Cloning Gibson assembly method Single reaction, can assemble...Central PMCID: PMC2574415. 3. Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA, Smith HO.Enzymatic assembly...terminus or the 3’ terminus of the ligation product. Comparison to other high throughput cloning strategies There... identical polyadenylation signals or promoters Gibson et al., 2009 MXS-Chaining, BioBricks, Bglbricks...Sladitschek and Neveu, 2015; Shetty et al., 2008; Anderson et al., 2010 Among the chaining-based methods...Additional Resources on the Addgene Blog Read about Gibson Assembly Learn About Other Plasmid Cloning Techniques... Find Plasmid Cloning Protocols References 1. Anderson JC, Dueber JE, Leguia M, Wu GC, Goler JA, Arkin... -
CRISPR 101: Multiplex Expression of gRNAs
TypeBlog PostPublishedSept. 10, 2020, 11:45 a.m.... you want to make will happen. There are many reasons to multiplex gRNAs, some being: Using dual nickases...the Bullock lab, and gRNAs can be inserted using Gibson Assembly or SLIC cloning methods. A BsaI-based ... up your multiplexed gRNAs with Golden Gate and Gibson assembly methods If you want to scale up the number...use some assembly methods such as Golden Gate or Gibson assembly. Golden Gate Assembly of gRNAs Golden ...supply it with another plasmid. Figure 3: Comparison of Multiplex Strategies including Standard PolIII-gRNA...Multiplexing in plants Qi-Jun Chen Lab Golden Gate/Gibson Assembly Multiplexing Plasmids: These plasmids ...you to assemble 2-4 gRNAs through Golden Gate or Gibson Assembly. gRNAs are inserted into the pCBC vectors... -
Antibodies 101: Fc Effector Functions
TypeBlog PostPublishedMay 18, 2023, 1:15 p.m....process known as opsonization. In antibody-dependent complement deposition (ADCD), opsonization of IgG and ...isotype class or even subclass, and are part of the reason why different isotypes drive different types of... in the pathogen’s cell membrane (Dunkelberger & Song, 2010). As cells cannot survive when holes are punched...(ADCC) is a slightly more direct method than opsonization. In ADCC, IgG antibodies bind to a pathogen ..., our antibodies have induced “stabbing” and “poisoning,” but let’s be honest, the elimination style the...10.1038/74704. PMID: 10742152. Dunkelberger, J., Song, WC. Complement and its role in innate and adaptive... Rachel Leeson...