...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...

... 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...

...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...

...Alyssa Cecchetelli  Listen to the C. elegans RNAi podcast segment RNA interference (RNAi) is extensively ...Eric Perkins  Listen to the TurboID and miniTurbo podcast segment A fundamental approach to studying membranes...by Izzy Mueller  Listen to the viral detection podcast segment As the primary cause for both the stomach... the absence of the target RNA sequence (in this case, norovirus RNA), the ribosome binding site is inaccessible...gene on the toehold switch to be expressed. In this case, the gene encodes the LacZ𝛼 protein. Once expressed...contributed by Michael Lemieux  Listen to the SH3 domain podcast segment The human proteome contains a breadth of...

...editing technique that relies on a fusion enzyme of Cas9 nickase + reverse transcriptase and a prime editing...., 2019), disrupting its canonical structure and Cas9 binding (Nishimasu et al., 2014). Figure...pegRNAs that use other bases in this location bind to Cas9 with canonical sgRNA structure (right). Created ...along with your intended edit. This prevents the Cas9 nickase from re-binding and nicking the newly synthesized...Zhang, F., & Nureki, O. (2014). Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA. Cell, ...doi.org/10.1016/j.cell.2014.02.001 Yan, J., Oyler-Castrillo, P., Ravisankar, P., Ward, C. C., Levesque, S....

...from the Zhang lab expressing S. pyogenes Cas9 and a gRNA cassette. It can be used to produce a ~10-fold ...and selection marker removal. CRISPR technology pdCas9-humanized (Plasmid #44246) Expression of a catalytically...catalytically inactive (dead), human codon-optimized Cas9 under the control of Murine Stem Cell retrovirus...carries CRE recombinase and a puromycin resistance cassette. No time to produce virus? Get the ready-to-use... (shRNA, miR-30-shRNA), mutation/editing (CRISPR-Cas9) or deletion (Cre), together with expression of ...

... the ability to correctly assemble a lacI/lacZ cassette (designed by NEB for use in optimization of assembly...sequence for beta-galactosidase in the lacI/lacZ cassette. Additional confirmation of accurate assemblies...colonies showed a mixture of mis-assemblies and very occasional uncut or cut/re-ligated pGGA destination plasmids...12- and 24-fragment assemblies of the lacI/lacZ cassette, and illustrates how the volume of the 1 ml outgrowth...and assembly efficiency Five fragment lacI/lacZ cassette assembly was easily achievable with high levels... 12- and 24-fragment versions of the lacI/lacZ cassette were designed and synthesized. In conjunction ...

...from single or multiplex CRISPR-Cas9 double-strand DNA breaks using SpCas9. More features will be added ...Currently, ICE is configured for Cas9 fromStreptococcus pyogenes (SpCas9). ICE Score - The editing efficiency...topic page xCas9: Engineering a CRISPR Variant with Pam Flexibility RNA Editing with Cas13 and REPAIR ...

...In today’s podcast, we sit down with Wei Leong Chew, a researcher at the Genome Institute of Singapore... Listen to the interview with Wei Leong Chew! Podcast breakdown Intro: Wei Leong Chew's career path and...the audio engineer and producer for the Addgene Podcast.      Additional Resources from the Addgene Blog...featured topic page Check out our Hot Plasmids Podcast Listen to our interview with synthetic biology ... addgene podcast...

...Sapozhnikov tells us how to use dCas9 (with no modifications!) as a CRISPR/dCas9-based tool for DNA demethylation...-red, a bright red pigment reporter for plants.  Cas7-11 and PASTE: Omar Abudayyeh and Jonathan Gootenberg... worked with us to share their new CRISPR tools, Cas7-11, a low-toxicity RNA editing tool, and PASTE, ...ancestral bias in CRISPR guide library design.  dCas9-directed DNA Demethylation: Guest blogger Daniel...

... hampered by the fact that even small Cas9 homologs like SaCas9 and their requisite gRNAs take up essentially...based system, the efficiency of gene editing is occasionally less than 1%, and virtually never exceeds 40%...et al 2017 use a dual vector system composed of SaCas9 and a gRNA in one AAV and a repair template in ...: PMC3277219. 6. Ohmori, Tsukasa, et al. "CRISPR/Cas9-mediated genome editing via postnatal administration...Porteus. "CRISPR-mediated integration of large gene cassettes using AAV donor vectors." Cell reports 20.3 (2017...

...interview and happy listening! Download the podcast here! Podcast breakdown (Time : Topic) 0:00 - 1:25 : Introduction...In the third installment in our podcast series, we chat with new Addgene Board Member, Michael Koeris...the audio engineer and producer for the Addgene Podcast.     Additional Resources Visit the Sample 6 website...the Addgene Blog Check the Other Posts in Our Podcast Series Get Additional Startup Advice Learn How ...