...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. ...

...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...

...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...

...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...

...used for each gene. At the moment, 13 full genome SpCas9 knockout libraries designed by different academic...Library, for example related to strategies beyond SpCas9, or species not currently included in Green Listed...libraries. As of now I have focused on human and mouse SpCas9 knockout libraries, but there are of course great...activity and minimize off-target effects of CRISPR-Cas9." Nature biotechnology 34.2 (2016): 184-191. PubMed...PMC5445660. 7. Chari, Raj, et al. "Unraveling CRISPR-Cas9 genome engineering parameters via a library-on-library...

...Optogenetic Tools Like pDawn  Making cassava plants resistant to cassava mosaic virus Dr. Fiona Robertson,...and Biotechnology expert. In order to preserve Cassava yields and to increase the potential for this drought...plant, Dr. Robertson aims to make use of the CRISPR/Cas9 technology to engineer plants that are able to destroy...

...including several pooled libraries: Cas13X and Cas13Y, two compact Cas13 proteins found in the metagenome...end-point sequencing assays. These plasmids use “Cas9-mediated adenosine transient reporter for editing...

...preps for the delivery of Cas9 endonuclease, Cas9 nickase, Cas9 activators, Cas9 repressors, select gRNAs...

...repository use Cas9 to cut DNA, Addgene Science Communications Intern Mary Gearing explains how Cas9 homolog,... suggestions. 8. A Match Made in Heaven: CRISPR/Cas9 and AAV Adeno-associated viral (AAV) vectors are...Validating Your Genome Edit Once you’ve used CRISPR/Cas9 to make a change to the genome, you need to check...

...pluripotent stem cell (iPS) technology and CRISPR-Cas9 constructs from Addgene to gene-edit mutations into...looking at a bunch of different strategies with CRISPR-Cas9. There are so many different variants and strategies... of those mutations, they now plan to use CRISPR-Cas9 technology to create cell lines and/or mouse models...ve proposed to use plasmids with modified nickase Cas9 protein in order to target the specific gene region...

...targeting, with a leg up on the alternatives - RNAi and Cas13. CRISPR-Csm doesn’t degrade proximal RNAs nor does...levels of off-target effects, both of happen with Cas13. CRISPR-Csm degrades both nuclear and cytoplasmic...nuclear-RNA targeting, where siRNAs are ineffective and Cas13 has steep off-target costs. Additionally, this system...imaging tool.   Fig. 1: Csm, shRNA, and Cas13 were compared head-to-head for gene expression changes...

...Producing yeast expression cassettes Traditionally, generating expression cassettes for yeast transformation...Cloning sgRNAs Production of Yeast Expression Cassettes Generating constructs with multiple point mutations...regions of homology and inserted into a linearized cassette, ready for transformation into yeast that same...

...Scientists SHOULD Be Using Twitter" podcast from Hello PhD In this podcast the hosts, Josh and Dan, discuss...monitoring that conference via social media. The podcast also features an interview with Dr. Stephani Page...and offers for postdoc positions. Check out the podcast for more tips on using Twitter as a scientist. ...

... absence of a protein, a yes-no plot is the best-case result. For example, you have introduced a GFP protein... not be as obvious in a dot plot. If that is the case for you, switching to a contour plot should indicate...interest. Created with biorender.com.   In such cases, instead of trying to quantify based on percentage...a good statistic to compare between samples. In cases where there is no clear distinction between the ...

.... Expert reviews on blog posts are done on a case-by-case basis, as many of our blog contributors primarily...webinars, or use other research methods. In the case of the antibody guide, primary research and previously...changes and require updating or retiring. CRISPR/Cas9, for example, has gone through rapid change in the...