...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...

...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...

...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...

... 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...

...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. ...

...positively and negatively controls the output. In this case, plasmid copy number is the input and gene expression...and keeping its expression level constant. In the case of the stabilized promoters from Segall-Shapiro ...varied in size or carried different genes. In these cases, gene expression also remained stable regardless...shuttling of the intermediates into other pathways. In cases where a specific ratio of products is needed, such...

...particular plasmid you are working with, but, in most cases at Addgene, we check what we call the “insert,” ...fusions, and multiple cloning sites. In the simplest case we have a standard backbone with one insert, and...and identity of the insert are correct. In other cases, we deal with multiple inserts or internal mutations...than two sequencing reactions. In the more complex cases, we sometimes have to design new primers to sequence...

...experiments in specific cell populations. Listen to Our Podcast Interview with Connie Cepko Combining Cre and GFP...recombination at LoxP sites. Placing a lox-STOP-lox cassette upstream of a gene of interest blocks transcription...absent. When Cre is present, it removes the STOP cassette, activating gene expression. Although Cre isn’...versions of non-modular proteins like Cre. Could Cas9-DOG be developed soon to allow genome modification...

...contributed by Brook Pyhtila  Listen to the CRY2clust podcast segment The Won Do Heo lab at the Korea Advanced...contributed by Meghan Rego  Listen to the soCoChR podcast segment Optogenetic stimulation is a powerful tool... Listen to the synthetic decaffeination operon podcast segment With the help of engineered microbes, used...FLIM-FRET acceptors  Listen to the FLIM-FRET acceptor podcast segment Article contributed by Christina Mork The...

...Many thanks to our guest blogger Juliet Moncaster! Juliet Moncaster is an Assistant Professor at the Boston... post was contributed by guest blogger Juliet Moncaster Leadership skills are amongst the professional...gain such experience? Your institution may offer occasional workshops on leadership and management skills...

...antibiotic resistance selection markers, gRNAs, and dCas9 proteins. Second, it can be introduced into a broader...and a plasmid that contains the Mobile CRISPRi cassette flanked by left and right Tn7 end sequences. Donor...pneumoniae, and P. aeruginosa) in addition to Vicrio casei, a bacteria isolated from cheese. Knockdown efficiency...promoters, selection markers, and different versions of dCas9. Following integration, Mobile-CRISPRi is stably...

...unusual blue-colored protein from the jellyfish Cassiopea xamachana (Blanquet and Phelan, 1987). Eight years.... Thus chromoproteins can be used as markers in cases where cost and resources are limited or for bioart... varied greatly between chromoproteins. In some cases, cells containing high-copy plasmid-borne chromoproteins...mesogleal protein from the mangrove jellyfish Cassiopea xamachana. Marine Biology 94:423–430 . https:/...

...systems, these systems remain valuable, especially in cases where CRISPR cannot be used (e.g. there are no suitable...targeted gene. Upon recombination, the GFP/NeoR cassette is inserted in place of exon 2. Thus, the targeted...use the Cre/Lox system described earlier. In this case, you design your targeting vector such that a set...sequence flanked by LoxP sites while the resistance cassette has been placed into an intron.    Figure...

...tested four serotypes containing an expression cassette with an ubiquitous promoter to express GFP in ...in a group of young rats. We found that, in this case, AAV1 (23%) and AAV6 (<5%, data not shown) were not...Addgene has shown me this was far from an isolated case. Our scientific support team answers hundreds of...But, especially for AAV data, that may not be the case. We can all acknowledge that negative data is something...