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... Listen here! Podcast breakdown (Time : Topic) 0:00 - 2:00 : Introduction to the podcast from producers...Addgene Podcast.     Additional Resources on the Addgene Blog Learn More About Alex's Work with Cas9 Activators...In this episode of the Addgene podcast, we sit down with Alex Chavez, a Clinical Fellow in Pathology ...Engineering. Alex is the first MD PhD we've had on the podcast, has worked extensively with CRISPR, and recently...Advisory Board with a particular interest in generating Cas9 based tools to enable facile control of DNA and ...

... had a large genetic footprint (two cassettes + a booster cassette), their expression heterogeneity required...integratable cassettes, both with doxycycline inducible promoters (Fig. 2a). The first cassette contains ...control. We also added the toxin-antitoxin stability cassette Axe-Txe so that the plasmid could be maintained... that Anabaena mARGs did not require a booster cassette, either, allowing us to reduce the genetic footprint...structural gene gvpA marked with EBFP2 and the second cassette contains the P2A-linked accessory genes gvpNJKFGWV...engineering a cell line, cotransfect the mARG cassettes 2:1 (A:N-V) with PiggyBac transposase and outgrow...

... CHIME works through transduction of Cas9-expressing (Rosa26-Cas9) hematopoietic stem cells with a lentiviral...genes pXPR_219 Violet-excited GFP (vex) Rosa26-Cas9 I-CHIME Inducible knockout of a single gene...Violet-excited GFP (vex) Rosa26-FlpO-ERT2; H11-Cas9 L-CHIME Knocks out a single gene in defined... Violet-excited GFP (vex) Lineage-Cre; Rosa26-Cas9 S-CHIME Knocks out pairs of genes sequentially...Violet-excited GFP (vex) Rosa26-FlpO-ERT2; H11-Cas9 Table 1: X-CHIME systems Proof of Concept These...Haining, W. N., & Sharpe, A. H. (2019). A CRISPR-Cas9 delivery system for in vivo screening of genes in...

...critical for Cas9 cleavage), thereby rendering the resulting construct resistant to Cas9 induced cleavage...can be used to identify Cas9/CRISPR-induced genome modifications. Occasionally, multimerization of the...through homology-directed repair (HDR) of a CRISPR/Cas9 induced double-stranded break. This site-directed... the repair, the template should be resistant to Cas9 cleavage. If a plasmid contains the template, site-directed...with the function of the resulting construct. In cases where the functionally important regions span several...plasmid (or other). This is particularly relevant in cases where a large plasmid with multiple functional regions...

...side of a stop cassette, upstream of an effector gene. In cells where the stop cassette is removed during...cells will express the effector or retain the stop cassette following recombination. Image from Isaacman-Beck...effector is expressed. In cells where the stop cassette remains, the effector is not expressed and the...attP sites to drive expression of either a stop cassette or an effector gene. HDVR (2X Hepatitis Delta ... Truncating the attP site upstream of the stop cassette results in decreased efficiency of recombination...preferentially leads to gene constructs where the stop cassette remains following recombination. This strategy...

...report. A scientist working in China was using CRISPR/Cas9 in an attempt to create gene-edited babies. Several...valid critiques being offered of He’s work: CRISPR/Cas9 genome editing is not far enough along, scientifically...research published on December 13 shows us, CRISPR/Cas9 techniques are continually being refined; it is ...an embryo—even if it’s not necessarily via CRISPR/Cas9. This leaves us with the second critique. Absent...agreed-upon international moratorium on utilizing CRISPR/Cas9 in humans. This topic was to be revisited at meetings...emerging technology. We could do this for CRISPR/Cas9 – or any other germline editing technique—too. As...not “what is the translational pathway for CRISPR/Cas9 research” but whether or not we will stop shuffling...

...sequencing in selected control and CRISPR-Cas9 injected embryos. In all cases, they did not observe off-target ... samples. Ma et al. designed plasmids containing Cas9 and two different gRNAs, and electroporated them... in editing between iPS cells and embryos make a case for conducting research in embryos should our society... decide that germline editing is permissible in cases of disease prevention. However, it is important ...alternatives are applicable in many situations. In the case of hypertrophic cardiomyopathy and other genetic... in increasing viable embryos, especially in the case of older mothers, for whom egg retrieval often results...

...single-percentage gel, you will have the option to cast your own gel. Handcast gels are significantly cheaper and generate...conditions and will cover most western blot use cases. If you’re running native (non-reduced) proteins...Tris-glycine 100 V, 1–2 hours Easy and cheap to handcast Short shelf-life; can alter proteins due to ...250 kDa Different sizes 4–20% gradient   Handcast or premade If you need a gradient gel, the decision...already made: gradients are extremely difficult to handcast, so unless you have an expert around, you’ll likely...hazardous chemicals, and are not always consistent. Precast gels, while expensive, reduce protocol time, can... CCD camera High initial (purchase) cost Precast gel Increased cost Save money Polyclonal...

...science communication. Science in the News hosts a podcast, seminars, a blog, conferences, and more for non-scientists...the interview with Science in the News, Part 1 Podcast breakdown Introduction to Vini, Amy, and SITN ...Approach Different Audiences Listen to the SITN Podcast Many thanks to our guests, Vini Mani and Amy Gilson...of Science in the News, and member of the SITN podcast team.     Amy Gilson recently received her PhD ...Science in the News, and is a member of the SITN podcast team. You can follow Amy on twitter @aigilson. ...the audio engineer and producer for the Addgene Podcast.     Additional Resources on the Addgene Blog ...

... gene was disrupted and silenced by a Lox-Stop cassette, resulting in the neurological deficits observed...Restoring the MECP2 gene by excision of the Stop cassette using tamoxifen-inducible Cre not only halted ...MECP2 protein, showing mutations corrected by CRISPR/Cas9 in wild-type human iPSCs. Image modified from Le...Le et al., 2019. Gene editing via CRISPR/Cas9 and RNA-mediated correction of MECP2 mutant alleles,... Klaus Rajewsky’s group recently used the CRISPR/Cas9 system to both precisely insert the MECP2-R270X ...Joung, PhD, and Feng Zhang, PhD, is pursuing CRISPR/Cas9 technology for precise correction of Rett syndrome...Rajewsky K, Chu VT (2019) Efficient and Precise CRISPR/Cas9-Mediated MECP2 Modifications in Human-Induced Pluripotent...