...Silvana Konermann, the first generation of CRISPR-Cas9 plasmids were not designed with gene activation ...question for her then was this: Can you engineer CRISPR-Cas9 activators that work well enough on any gene that...describe just such a system, which they call CRISPR/Cas9 Synergistic Activation Mediator or SAM. SAM is an... three components: 1. A nucleolytically inactive Cas9-VP64 fusion; 2. A sgRNA incorporating two MS2 RNA...system, Konermann explained, was new knowledge about Cas9’s crystal structure. “I thought maybe we could use...The activator (CRISPRa) sgRNA library uses the sunCas9 system and contains 10 sgRNAs for each transcription...levels of control in the system. First of all, the Cas9s can be turned on and off, to reversibly activate...

... kit to CRISPR/Cas9? When would you choose one or the other? Musunuru: The CRISPR/Cas9 system is easier...system for CRISPR/Cas9 (also available from Addgene) only has two plasmids. In many cases, CRISPR may be ...makes possible, and how it compares to his CRISPR/Cas9 system. Addgene: For people unfamiliar with this...two ends and put them back together, there are occasional errors. That can be advantageous if you are trying...genes that have been implicated in disease or, in cases where particular variants or mutations have been...more efficient. You might often start with CRISPR/Cas9, but TALENs do have more flexibility in terms of...to date suggest TALENs may be cleaner than CRISPR/Cas9. Reference: Quirong Ding et al. "A TALEN genome-editing...

...CRISPR/Cas9-based systems have been developed for generating knockin mice but because CRISPR/Cas9 engineering... of a stop cassette upstream of the target gene in the targeting vector. The stop cassette is floxed, ... arms that flank the gene of interest. In either case, a positive selection marker such as an antibiotic...recombination for precise gene insertion. However, CRISPR/Cas9 systems are more useful for generating knockout ...to engineer knockout mice. ZFN, TALEN, and CRISPR/Cas9 systems can all be targeted to specific regions ...introduce multiple base pair deletions, but CRISPR/Cas9 can also introduce point mutations. While using ...so Cre-mediated recombination deletes the stop cassette, which permits expression of the target gene. ...

...Institute to provide lentiviruses for delivery of SpCas9 and Cas9 nickase for genome editing. You can also enhance...facilitated their fast adoption. However, in many cases the plasmids themselves are only the starting point...Like all things in science, getting a high titer Cas9 lentivirus prep takes a bit of time and effort that... makes it unuseable. With the large size of the SpCas9 nuclease (~4.2 kb) this is a particularly prominent...expression of your gene of interest with a variety of Cas9 activators available in lentiviral format. Finally... pathways. Find Validated gRNAs to use with your Cas9 Virus Pooled libraries now available in lentiviral...be used to infect a cell line already expressing Cas9. As John Doench (Broad Institute and member of the...

...? Check out Addgene's Lentiviral dCas9 SAM vector and Piggybac dCas9 SAM vector! On/off control all the...out the gene for your protein of interest. CRISPR-Cas9 and a custom gRNA is all you need to generate a ...interference (CRISPRi) targets a catalytically dead Cas9 fused to a transcriptional repressor to your gene...repression of protein expression. Addgene has a dCas9 KRAB plasmid for transient knockouts!  Turn it on...) are transcriptional activators fused to a dead Cas9. The system is led, via a custom gRNA, to your gene...protein levels. Looking for a DD? Check out our DD-Cas9 vector! The price of tunable control These systems...Plasmid 101: Repressible Promoters CRISPR 101: Which Cas9 Do I Choose for my Experiment Additional resources...

... 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 ...

... when compared to the most commonly used Cas9 variant, SpCas9. These properties, combined with other benefits...Generating Mouse Models Using CRISPR/Cas9 Cpf1 Update: Comparison to Cas9 and NgAgo Special Delivery: Fluorophore... or more additional posts that deserve a peek in case you missed them. July 2016 was a fantastic month...generate mouse models.   Cpf1 Update: Comparison to Cas9 and NgAgo As you might expect given the excitement...

... 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...

...resistance cassette and you aren’t introducing an additional antibiotic resistance cassette along with...Antibiotic selection Selection is straightforward in cases where the inserted DNA includes an antibiotic resistance...plasmid backbone contains a kanamycin resistance cassette, you would plate the cells on kanamycin-containing...carrying plasmids that contain a kanamycin resistance cassette will grow. However, often times, the plasmid backbone...with your gene of interest. In these cases, antibiotic selection will select for cells that have been transformed...

...bacterial selection cassette for growth and cloning in bacteria and a mammalian selection cassette to screen for...cells, you will not need a mammalian selection cassette (e.g. 1435 pSG5L Flag HA). You can also upgrade...in an isolated, controlled environment. In most cases bacterial expression vectors are inducible (e.g....with an integrating vector expressing a selection cassette with 5′ and 3′ flanking sequences matching the...fusion proteins. Pick your promoter, selection cassette and visualization vehicle to conduct your custom...

...RNA with Cas13a (C2c2) Maybe you’d like to avoid editing the genome and just stick to RNA? Cas13 may be ...Corn’s lab describe how a better understanding of Cas9 biophysics can help enhance HDR. CRISPR 101: Ribonucleoprotein...The PAM Requirement and Expanding CRISPR Beyond SpCas9 The closer your edit is to the PAM site, the higher...repair. Check out this post to see if an engineered Cas9 variant might work for your experiment. PITChing...tool to help you design, pick, and close guides for Cas9 and Cpf1. This tool supports over 250 genomes, and...

... all influenced by an understanding of Cas9 biophysics! Casilio: An Adaptive, Multitasking “CRISPR-OS”...Medicine described Casilio, a system that uses modular RNA binding proteins, dCas9, and a modified gRNA... the Casilio system works and how you can customize it for your experimental needs. Note: Casilio is no...