...post was originally written by Joel McDade and significantly updated in 2022 by Susanna Stroik.  The advent... than ever to make precise, targeted genome modifications. Cas9 has been modified to enable researchers...uses a “nicking” Cas9, in which one of Cas9’s two critical enzymatic residues has been converted to an alanine...instead.  Choosing a Cas9 for site-specific modifications Site-specific deletions and knockouts Some experiments...Perhaps you need to introduce a single nucleotide modification at a defined amino acid to mimic a patient mutation...

...Why pseudotype? Lentiviruses and retroviruses typically target immune cells with their wild-type envelopes...out this review article published in Reviews in Medical Virology (Li et a., 2018).   Figure 2:...The glycoprotein will be incorporated into the replicating rabies virus envelope, travel retrogradely, and... Pseudotypes: Precious Tools to Improve Gene Modification of Hematopoietic Cells for Research and Gene...Restriction of Transsynaptic Tracing from Single, Genetically Targeted Neurons. Neuron 53:639–647 . https:/...

...the Society for Advancement of Chicanos/Hispanics and Native Americans in Science’s (SACNAS) annual conference... written up in mSphere. A post-meeting survey indicated that the program was largely a success, but also... even more valuable program to Microbe 2020 in Chicago.  Finding our place in networking and mentoring... career scientists, students, and those from historically underrepresented groups. Through such mentoring...

...many cell types and often results in biallelic modifications. Most commonly, DSBs are repaired by the nonhomologous...combining these two strategies allows you to specifically incorporate homo- or heterozygous mutations....in the field at the time but had never been systematically analyzed: introducing silent CRISPR/Cas9 blocking...efficiently incorporated during HDR. We decided to systematically characterize this phenomenon and found a general...University. He currently works to understand pathological mechanisms of Alzheimer’s disease by developing...

...even when larger vector genomes are packaged, the physical size of the genomes is still ~4.7kb. But despite...overlapping strategy has a region of homology, typically ~400-1400 bp long between part A and B of the ...overlapping split vector had an improvement in muscle physiological performance.  2. Trans-splicing Overview: For...and may need to be optimized for each unique application (Trapani et al., 2015).  Expression of smaller..., was 81% of that from a single vector, but significantly greater than the expression from an overlapping...

...favorite twitter #agarart accounts, Petri dish Picasso (@PetriPicasso) came together and delivered very impressive...are some of our 2017 contest winners:   Chicagoland's Community Biology Lab (@ChiTownBio) and one ...Mackin Lab from University of Colorado -Anschutz Medical Campus (@CSD_CUAMC) caught a case of holiday cheer...

...simple in C. elegans and bacteria. But CRISPRing typical mouse models, including creating Cre-dependent ...on both sides and the T7 promoter on one end. Typically, Easi-CRISPR works best with insertions of 2 kb...models varies from 8.5-100% of pups born, with typical efficiencies of 30-60%. Quadros et al. attribute...2.5x more animals than i-GONAD and is a more technically difficult process. Overall, the main drawback... out our CRISPR Guide Find Plasmids for Cas9 Purification Find CRISPR Plasmids for Mammalian Expression...

...any molecular biologist Tips for protein purification Replica plating Anti-CRISPR Retroviral vectors Career...source of information on recent advances in the biological sciences and techniques that simplify and expedite... help hone your blog post for our audience of biological scientists from a variety of different fields...

...Investigator III at Novartis, a multinational pharmaceutical company based in Switzerland. After completing...completing her PhD in Chemistry and Chemical Engineering with Harry Gray at Caltech, Crystal moved on to a ...Chapter 7: How Crystal developed her science communication skills and how they've helped her at Novartis...scientific teams at the Novartis Institutes for BioMedical Research to develop novel biologics for the treatment...

...Addgene podcast, we sit down with Alex Chavez, a Clinical Fellow in Pathology at Masachusetts General Hospital...Church and Collins labs at the Wyss institute for Biologically Inspired Engineering. Alex is the first MD PhD...and explains why he loved the first 2 years of medical school. 7:05 - 10:55 : Alex's focus during graduate...postdoc 12:01 - 13:21 : The Wyss Institute for Biologically Inspired Engineering and why Alex decided to...

...acquires through Addgene to produce molecular biological tools like Taq polymerase, T7 polymerase, EcoRI...scientists to continue to do excellent work in the biological sciences. Future awardees Now that this first...concluded, Addgene and Seeding Labs will be opening applications for a second round of plasmid awards on June...excited to discover what new and creative ideas applicants will propose for plasmids from the Addgene community...

...infectious disease once limited to small pockets in Africa has spread to urban areas and across the ocean....percent of the most recent cases, primarily in West African countries. Even that grim estimate might prove ...protein present on the virion surface – making it critical for viral attachment and fusion. Once inside a...glycoprotein’s role in infection also makes it a critical component of vaccines and the target of neutralizing...

...analyzing your cells using flow cytometry, you are typically measuring the presence or absence of certain markers...reporter, such as GFP or mCherry, through genetic modification. As your protein of interest will only occur...free amines on and inside the cells and are thus typically used for distinguishing live from dead cells. ...describe a basic panel (Table 2, Figure 5) for a hypothetical experiment in the lab. Let's say I am culturing...Associate in the lab of Arlene Sharpe at Harvard Medical School. His projects include investigating negative...

...the user. This approach is useful for several applications as will be discussed below. The software could...has a maximum of one gene knocked out. In this genetically heterogeneous population, cells behaving differently... you could majorly accelerate progression into clinical trials. Secondary screens  If you have generated...genes you’d like to test for phenotypic effects. Typically the phenotype would be studied in vitro, but there...contain multiple gRNAs targeting all genes in the indicated species and were suggested to be the best gRNAs...

...Antibody characterization is critical to enhance reproducibility in biomedical research. eLife, 13, e100211...tell only part of the story. This year, we made significant strides in making science more accessible and...launched AAV Packaged on Request, a service that dramatically expands and simplifies your viral vector options...

...promoter. It can be used to drive Cre expression specifically in neurons. AAV Delivered Cre - A useful tool...Indeed, this AAV provides you with a means to specifically activate expression of any Cre-dependent gene...pmSyn1-EBFP-Cre Of course there are also more complicated ways to use this particular AAV. For instance...Additionally, you could use the retrograde form to specifically express Cre in neurons with axons that extend...

...service. A small subset of AAV plasmids will have technical or legal restrictions that make them ineligible...be reviewed by Addgene for legal approvals and technical compatibility with our production process. This...request is approved, you will receive an email notification that your request is ready to order. An “Add...found in our in-stock preps. If a request has technical considerations that may affect expected titers...

...incredibly useful molecular biology tool, but like any biological system, it has certain drawbacks. First, the ...Gerald Rubin’s lab sought to make complex genome modifications in Drosophila using multiple recombinases. To...offspring carried both the recombinase and GAL4, indicating that the recombinases were inherited normally...Chinese hamster ovary (CHO) cells, as well as in cortical neurons in layer 2/3 of the mouse brain. Rubin...

...  “This enables you to do customized genetic modification on a scale that was really not possible before...genome engineering with CRISPRs was limited to modification of a few loci at a time,” Wang said. “It was... lentiviral libraries and their demonstrated application offer proof-of-principle that CRISPR-Cas9 can...aimed for 300 cells per construct, but in some biological systems one “might need larger numbers.” Further...

...: Find sequences for plasmids assembled from publications and commercially available sources, as well ... as expression patterns, post-translational modifications and polymorphisms Codon optimization tool: To...making it ready to copy and paste into other applications Primer design tools Many of the above-listed... well as primer design for different cloning applications. Primer3 and Primer3Plus: For general primer...