...and Harvard Medical School. Scientists making transgenic organisms with Cas9 should be aware of the potential...conducted with extreme caution. Why does making transgenic organisms with Cas9 risk creating a gene drive...drives that can spread through populations of transgenic laboratory organisms but not wild organisms. ...targeting a synthetic sequence will only spread in transgenic laboratory populations with that sequence2. Both...collective technologies are developed safely and transparently.  For more information, visit www.sculptingevolution.org...

...The goal of Kiran Musunuru's lab in Harvard's Department of Stem Cell and Regenerative Biology is to ...modeling work in human pluripotent stem cells: a transcription activator-like effector nuclease (TALEN) kit... designer proteins that will introduce a double-strand break in the genome anywhere you would like. Cells...cells after the plasmids have been introduced by transfection or electroporation. All you do to create a protein...genetic backgrounds. It becomes more of an apples-to-oranges comparison. Addgene: Can you compare the TALEN ...the target site. Also, any time you create double-strand breaks, there is a possibility of introducing a...

...type VI-D enzymes are the latest tools in the transcriptome engineering toolbox. How were Cas13d enzymes...small hairpin RNA interference, dCas9-mediated transcriptional inhibition (CRISPRi), and Cas13a/Cas13b RNA...only time will tell how large the genome and transcriptome engineering toolbox will be. It is, however,...community. References Konermann, Silvana, et al. “Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR...Editing with REPAIR Get tips for using Cas9 Transcriptional Activators Learn about Cas13a (C2c2) Characterization...

...19329793. PubMed Central PMCID: PMC2719376. 5. Barany, Francis. "Genetic disease detection and DNA amplification...1986365. PubMed Central PMCID: PMC50775. 6. Barany, Francis, and David H. Gelfand. "Cloning, overexpression...Central PMCID: PMC208194. 8. Luo, Jianying, and Francis Barany. "Identification of essential residues in Thermus...(1989): 245-254. PubMed PMID: 2753355. 12. Barany, Francis. "The ligase chain reaction in a PCR world....25. Luo, Jianying, Donald E. Bergstrom, and Francis Barany. "Improving the fidelity of Thermus thermophilus...temperatures (up to 95 °C) and is active over a range of elevated temperatures (37–75 °C). T4 DNA ligase...temperature near the melting temperature (Tm) of the DNA strands. This selectively reduces the concentration of ...

...fused to a fluorescent protein. “Frankenbody,” like the infamous frankenstein, was constructed by grafting...called this probe the HA frankenbody (Zhao et al., 2019).  To develop HA frankenbody, the Stasevich lab tested...can you do with the HA frankenbody? Another large advantage of the HA frankenbody is that it remains bound...also proved that HA frankenbody can be effectively used to track single mRNA translation in U2OS cells. HA...acids, thus it is translated quickly by the ribosome. This feature provides HA frankenbody the ability to...properly once inside cells.  Developing the HA frankenbody To combat the issues with these antibody based...lab observed colocalization of GFP tagged HA frankenbody with an mCherry, 4xHA tagged histone protein ...

... successfully mediated transduction and expression of the full-length transgene in vitro and in vivo (...this, larger functional transgene products were produced following transduction.  So what’s going on here...between part A and B of the transgene to reconstitute the full-length transgene (Chamberlain et al., 2016...cells in mice. This vector transduced ~10% of cells, similar to the transduction rate of a single AAV. Odom...muscle physiological performance.  2. Trans-splicing Overview: For trans-plicing, splice site donor and acceptor...reconstitute the two pieces of the transgene.The AAV encoding part A of the transgene contains a promoter and the...fragment of the transgene, followed by a splice site donor. The AAV encoding part B of the transgene contains...

...phosphatase-accessible. Kinase Translocation Reporters (KTRs) Kinase Translocation Reporters (KTRs) are genetically...particularly important for regulating signal transduction and measuring kinase activity at the single-...provides an alternative tool with their Kinase Translocation Reporters (KTRs) whose cellular localization...activity is enhanced, leading to cytoplasmic translocation of the reporter. As a result, the KTR is shuttled...nuclear export and import rates can influence KTR translocation. Work from the Covert lab suggests that these... negligible effect on KTRs, however nuclear translocation rates are another variable to consider when ...measurements of kinase activity in single cells using translocation reporters. Nature protocols, 13 1, 155-169. ...

... recent works, transdifferentiation is usually carried out by expressing transcription factors specific...successfully transdifferentiated into neurons and β-cells [40-42]. Reprogramming and transdifferentiation: When...redifferentiated to a different cell type. Transdifferentiation In this process, differentiated adult somatic...plasmid electroporation (episomal) [7, 8] and mRNA transfection [9]. Many of the plasmids used for these methods...variety of plasmids for differentiation here. Transdifferentiation Dedifferentiation to an intermediate pluripotent...primitive pluripotent stem cell state, through transdifferentiation adult somatic cells are converted directly...newt over 100 years ago [36]. While natural transdifferentiation is rare in mammals, an example is observed...

...expressing a novel reverse transcriptase (RT), MarathonRT. Reverse transcriptases are critical tools for ... to transform and express in bacteria, which, in turn, makes them more adaptable to a broad range of applications...!  MarathonRT: A highly processive reverse transcriptase Article contributed by Will Arnold  Listen ...RTs in this regard and is even able to reverse transcribe the extremely long, highly structured HCV genome...structured template!). MarathonRT also faithfully transcribes RNA to DNA with low error rates compared to other... are dependent on long, error-free, reverse transcription of RNA such as long-read cDNA sequencing and...29109157. Nanobody toolkit to monitor retrograde transport at the cell surface Article contributed by Marcy...

...Addgene-Seeding Labs Plasmid Grant Check Out Previous Addgene-Seeding Labs Plasmid Grant Awardees Listen to Our...Labs and Addgene. The Addgene-Seeding Labs plasmid grant is a collaborative endeavor providing plasmids to...who were awarded the Addgene-Seeding Labs Plasmid grant this year. These scientists work on projects spanning...and to increase the potential for this drought tolerant plant, Dr. Robertson aims to make use of the CRISPR... different CRISPR applications in plants.     Transgenic yeast as source of dietary supplements Dr. Mrinalini... proteins. In this project she aims to create transgenic yeast as an alternative source of essential omega... diverse groups of scientists working in a wide range of research fields and geographical locations. We...

...received a grant from the National Institutes of Health that will address this problem.The grant, which is...within the next year. NIH grants and Addgene’s mission This is the first NIH grant that Addgene has received... of California Davis School of Medicine on this grant. Dr. Trimmer has created thousands of high-quality...NIH’s vision and support for this project. This grant will allow Addgene to further our mission of scientific...working with the community to create a fully transparent resource where researchers can easily share, ...project will be supported by a NIH BRAIN Initiative grant (NS119916). The content of this article is solely...P, Frenzel A, Wong J, Koh XY, Eng H-Y, Lane D, Lefranc M-P, Clark M, Dübel S (2018) When monoclonal antibodies...

...plasmid page here). Now I spend much less time wrangling bacteria and instead help Addgene fulfill its ...better and make scientists’ lives easier. Why transition to Addgene? So why did I join Addgene? Was I ...easier. I’m hoping my story demonstrates that transitioning from academia doesn’t have to mean abandoning...management, information distillation, etc) are transferable to many different fields. If you start thinking...

...fluorescent protein transcription in response to river contaminants and transform these plasmids into ...Addgene-Seeding Labs Plasmid Grant Check Out Addgene-Seeding Labs Plasmid Grant Awardees in 2016 and 2017... specifically, the Addgene-Seeding Labs plasmid grant provide plasmids to these researchers. In the third...third round of the Addgene-Seeding Labs plasmid grant we are excited to introduce the 5 awardees and their...into a bacterial strain. The sensor will be transported inside a small 3D printed robot that will “swim” ... diverse groups of scientists working in a wide range of research fields and geographical locations. In...see how all of the Addgene-Seeding Labs Plasmid Grant awardees' projects progress. Subscribe to the Addgene...

...course much more difficult than in cell culture, but Ran et al. have shown that CRISPR and AAV can be used...Thakore PI, Moreb EA, Rivera RM, Madhavan S, Pan X, Ran FA, Yan WX, Asokan A, Zhang F, Duan D, Gersbach CA...WL, Widrick JJ, Yan WX, Maesner C, Wu EY, Xiao R, Ran FA, Cong L, Zhang F, Vandenberghe LH, Church GM, ...: 25692716. PubMed Central PMCID: PMC4335351. 9. Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz...) to remove mutated exons from the dystrophin transcript. Unfortunately, the oligonucleotides only modestly...in one experiment, they found the exon-skipped transcript constituted 59% of total dystrophin mRNA, similar...low to no off-target activity at the ten highest-ranking predicted off-target sites. Each paper characterizes...

...874 . https://doi.org/10.1038/nmeth.3993  Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu.../doi.org/10.1038/s41586-019-0908-x  Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, Ishitani...949. https://doi.org/10.1016/j.cell.2014.02.001  Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz...gene delivery due to their low immunogenicity and range of serotypes allowing preferential infection of ...prime editors. While using AAV to deliver large transgenes might seem daunting, it’s actually a challenge...split AAVs. In general split AAVs break a large transgene in two pieces and package each piece into an individual... individual AAV. When a cell is transduced by both AAVs, the full length gene and/or protein is reconstituted...

... two of the remaining viral proteins. Only Nsp11 ran bigger than expected, and Orf7b gave a lot of background...received so many requests (175 and counting) that we ran out of FedEx supplies at one point and had to go ...right) from the University of California, San Francisco. Manon Eckhardt and Melanie Brewer are on the ...

...For the last 20 years, I was a patent counsel and ran a group at two fairly large companies, Genzyme and...What is your day-to-day like? BN: I've made the transition now to being general counsel of a small start-up...

...limitations of the Cas9 complex," said study author F. Ann Ran, a graduate student in Feng Zhang's lab, in a press..., in the labs of Chase L. Beisel and Rodolphe Barrangou, had applied the CRISPR/Cas9 system to developing...

... process. Julie is dedicated to volunteerism and ran the Mayor’s Midnight Sun Marathon in Anchorage, for...

... Publication describing them here. References 1.Ran, F. Ann, et al. "Genome engineering using the CRISPR-Cas9...plasmid post transfection. The 2A "self cleaving" peptide causes the ribosome to translate Cas9-2A and ...component of most CRISPR systems and generates double strange breaks at target sites 5' of the 5'NGG3' PAM sequence...backbone. This particular backbone can be used to transfect a variety of mammalian cells and can be modified...are some additional features. The first and most imporant is that, upstream of the Cbh promoter, this plasmid...can be used to test for SpCas9 expression post transfection by western blot. Overall, this is a versatile...