...? While properly designed perturbations – in the case of screens using CRISPR technology, gRNAs to knockout...formats: all-in-one (lentiCRISPRv2, expressing both Cas9 and an individual gRNA from this single vector) ...only an gRNA, for use with cells that already have Cas9). Each library consists of approximately 3,000 gRNAs...activity and minimize off-target effects of CRISPR-Cas9." Nature biotechnology (2016). PubMed PMID: 26780180...Learn to Conduct Genome-wide Screens with CRISPR/Cas9 Get John Doench's Tips on gRNA Design Check Out ...

...binding affinity (Janda et al., 2016; Torres & Casadevall, 2008) but…does it really do anything? Fc functions...an antigen-bound antibody and induce (or, in the case of the Fc receptors FcγRIIB1 and FcγRIIB, inhibit...system's classical complement pathway, starting a cascade of events that results in the pores being opened....2009.139 Janda, A., Bowen, A., Greenspan, N. S., & Casadevall, A. (2016). Ig Constant Region Effects on Variable...articles/10.3389/fmicb.2016.00022 Torres, M., & Casadevall, A. (2008). The immunoglobulin constant region...

...inactivates or completely removes (in the case of Gateway cloning) one cassette. Counterselection allows the scientist... Scientists introduce an antibiotic resistance cassette within the coding region of the gene they are ...is best practice not to use the same resistance cassette for the mutation and for plasmid selection. Additionally... vector typically has two separate resistance cassettes and your gene of interest is cloned into/inactivates...

... the flexibility needed for multiplexing. In the case above, you can’t costain with your desired antibodies...easily manipulated into more useful tools. In the case of isotype conversion, the variable regions of a... species and/or isotype (Liddell 2013). In most cases, the resulting chimeric antibody retains the binding...properties (Morelock 1994). While in the majority of cases the differences are subtle, chimeric antibodies ...when multiplexing with chimeric antibodies. In the case above you should use an anti-rat IgG1 secondary ...

...independence, such as museums, YouTube channels and podcasts, science pub nights, and pop sci books. Most scicomm... that is widely used but has a very specific use-case (definition) in a specific field.  For example, ...also students in AP biology classes Creating a podcast about genetics using dog breeds as an example ...interested in breeding dogs who also enjoy listening to podcasts A series of Tweets breaking down a new publication...others from accessing their work. But that’s not the case. In fact, it’s really the opposite. Unlike emails...

... PhD Podcast episode 15 "Simple tricks for time management: the pomodoro technique": This podcast talks... References and Resources You Can Grad School podcast episode 2 "Organizing Literature" : I found out...out about Zotero here Dear Grad Student podcast episode 2 "Essential Grad School Resources: Citation Managers...Managers, Programming, & Productivity": This podcast talks about Mendeley, Zotero, and other citation ...

... carefully presented case for better more diverse science. It's a series of case studies in what happens... other works of Dr. Sacks, Musicophilia includes case studies of relevant neurological atypicalities, ...the Dark Lord rules all, the books center around a cast of endearing characters who decide to take matters...Ice Age to the 1980s, Sarum follows an intriguing cast of characters from five different families over ...

... to help you find your desired plasmid. In many cases, you will need a screening strategy as well. In ...comes in. Let’s take a look at All_in_one_CRISPR/Cas9_LacZ, a CRISPR gRNA plasmid from Lynne Postovit’s lab..., without the need to prep your plasmid. In this case, you’d be using colony PCR to detect your insert...the other primer anneals to the backbone. In this case, if there is no insert in the plasmid, you won’t...order of nucleotides within a DNA molecule, in this case a plasmid. Sequencing is one of the surest ways ...

...selective degradation of FKBP12F36V-BRD4 using CRISPR/Cas9-mediated knock-in, and to evaluate in vivo degradation... and endogenous ENL was inactivated using CRISPR/Cas9 gene-editing. Featured molecules: dTAG-7 and dTAG...and endogenous MELK was inactivated using CRISPR/Cas9 gene-editing. Featured molecules: dTAG-7, dTAG-13...engineered to express YY1-FKBP12F36V using CRISPR/Cas9-mediated knock-in. Featured molecule: dTAG-47 Additional...Knock-In and Conditional Mouse Models CRISPR 101: Cas9 Nickase Design and Homology Directed Repair Plasmids...

...progestin hormone therapy resulted in 76,000 fewer cases of cardiovascular disease, 126,000 fewer breast ...methods of sex and gender analysis and 2) provides case studies with concrete examples of how gender analysis...analysis leads to discovery and innovation. One such case study looks at the effects of using all female cells...also consider the sex of the stem cells. In this case, they found that sex-matching of donor and recipient...analyze these factors during an experiment. In the case of stem cells, these factors may include cell type...

...activity and minimize off-target effects of CRISPR-Cas9." Nature biotechnology (2016). PubMed PMID: 26780180...transcriptional activation by an engineered CRISPR-Cas9 complex." Nature (2014). PubMed PMID: 25494202. ...PMC4559080. 8. Shalem, Ophir, et al. "Genome-scale CRISPR-Cas9 knockout screening in human cells." Science 343.6166..."Genetic screens in human cells using the CRISPR-Cas9 system." Science 343.6166 (2014): 80-84. PubMed ...Wong, Alan SL, et al. "Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM." Proceedings of the...

...Yoon, S., & Kim, H. H. (2019). SpCas9 activity prediction by DeepSpCas9, a deep learning–based model with...2023). This method uses a prime editor (typically a Cas9 H840A nickase fused to a reverse transcriptase) ... and PE5 prime editing strategies) based on a DeepSpCas9 score ranking (Kim et al., 2019). PRIDICT saves...Steensel, B. (2021). Impact of chromatin context on Cas9-induced DNA double-strand break repair pathway balance...

...noticing unusual cases of acute hepatitis in the pediatric population. Once ~400 cases had amassed in places...here it appears that the culprit for these unusual cases was either 1) a physiological effect of SARS-CoV...inactivation of the vector can still occur even in the case of no previous immunity existing for that adenoviral...References and resources References Bauzon, M., Castro, D., Karr, M., Hawkins, L. K., & Hermiston, T. ...

...inserted the deaminase into the C-terminal domain of Cas9, which narrowed the editing window and reduced A.... gGBE consists only of engineered MPG fused to nCas9. First, MPG removes the G base from DNA to create...site. Like in most other base editor designs, the Cas9 nicks the non-edited strand to encourage the cell... using the edited strand as a template — in this case, encouraging it to incorporate a new base across...2024; Ye et al., 2024; Yi et al., 2024). In each case, a UNG enzyme was modified to recognize T and/or...

...Blainey’s lab has developed a multiplexing approach to Cas9-based pooled CRISPR screens called CROPseq-multi...Anzalone et al., 2019, including modifications to the Cas9 or reverse transcriptase enzymes, inhibition of ... cell types at different genomic loci — in some cases, over 20-fold higher editing than PEmax. The authors...end of the pegRNA, which is not well protected by Cas9. This is similar to the proposed mechanism for engineered...of applications, including CRISPR activation by dCas9-VPR, prime editing, and delivery of disease-relevant...

...death. The team used this cassette to knock out genes, knock in a GFP cassette, and introduced site-directed...continually produce antitoxin to avoid cell death. In the case of plasmids, toxin-antitoxin systems act as “selfish...for Zeocin resistance (Figure 2). This MazF-ZeoR cassette is flanked by two direct repeats which can be ... selects for those cells that have removed the cassette through a second recombination event between the...

...fluorophore-conjugated antibody commercially available. In other cases, fluorophore-conjugated antibodies may exist, but...may want to look at other methods. In the above cases, a good alternative is using a two-protein detection... antibodies that block surface proteins. In that case, you would want to look at negative selection: labeling...+ T cells, B cells, and dendritic cells. In this case, we simply need to label the lineage markers of ... a separate fix/perm cytosolic procedure in that case.     Figure 3: For intracellular labeling...

... Today on the Addgene Podcast, we have another interview conducted by European Outreach Scientist Benoit... Listen to the interview with Joachim Goedhart Podcast breakdown Intro Chapter 1 - How Joachim ended up...the audio engineer and producer for the Addgene Podcast.      Additional Resources from the Addgene Blog... addgene podcast...

...still be taken when using them. There are suspected cases of insertional mutagenesis in humans (1) and AAVs...Currently, AAVs are being used to deliver CRISPR/Cas9 but have the potential to deliver other therapeutic...used with new exciting technologies (such as CRISPR/Cas9) as they allow for gene delivery in an efficient... 24086725. PubMed Central PMCID: PMC3785459. 4. Castle, Michael J., et al. "Adeno-associated virus serotypes...Expression in Mammals A Match Made in Heaven: CRISPR/Cas9 and AAV Viral Vector Elements Additional Resources...

...Today’s episode of the Addgene Podcast features guest host and Addgene European Outreach Scientist, Benoit...researcher. Listen to the interview with Tom Ellis Podcast breakdown Intro: How Tom ended up in academia and...the audio engineer and producer for the Addgene Podcast.      Additional Resources from the Addgene Blog... addgene podcast...