...(MP6) permits quick, large-scale mutation of the gene of interest. They created a selection phage (SP)...transcription from the accessory plasmid (AP) encoding phage gene III. The accessory plasmid library contained all... a library PAM, it would induce transcription of gene III, permitting phage propagation and keeping that...evolution to generate and select for Cas9 mutants with PAM flexibility. In this system, the mutagenesis plasmid...this paper are available at Addgene.  Additional Resources on the Addgene Blog Learn about Engineered...another 72 hours of phage-assisted evolution, generating xCas9 2.0-2.6. This second round of evolution... Cas9 variants with activity on non-NGG PAMs, generating xCas9 3.0-3.13. To test xCas9 3.0-3.13, Hu et...

...22,000 protein coding genes in the human genome with fluorescent proteins. For each gene, we needed to design...way that prevents re-cutting but does not disrupt gene expression. (See section below.) For guide design... Zuckerberg Initiative. We hereby announce the general availability of new a tool for CRISPR scientists...analyses, but it also allows you to download any generated info for your own purposes, or delete the originals...at existing tools. As has been written here on Addgene, there has been a Cambrian-like explosion of software...that it can be used in the rank equation above. Generally, the greater this distance between the cut and...sequence inserted by HDR. Although this practice is generally advised, there is little detail published on how...

...guides and an NgAgo expression plasmid to edit a gene in vivo. Try NgAgo for Yourself On the other hand...were having difficulty achieving indel formation (gene disruption) with this new technology. To dig more...asked if they could detect indels (an indication of gene disruption ability) with NgAgo, one respondent out... do not necessarily represent the views of Addgene. Addgene performs Sanger sequencing on select regions...the water. On the bright side, successful indel generation and epitope knockin was achieved by a few respondents...Twitter - Tweet suggestions and tips with #NgAgo The Addgene Blog - Leave suggestions and tips in the comments...comments section below or contact blog@addgene.org if you'd like to write a more detailed post It may take...

...directional gene transfer, analogous to traditional restriction enzyme cloning. Sticky-end mediated gene transfer...adeno-associated viral vectors) are commonly used for in vivo gene delivery due to their low immunogenicity and range... the potential “sticky end” transgene insertion mediated by Cpf1-generated overhangs. Exploring the efficiency...suited for genome editing.  Find Cpf1 Plasmids at Addgene   Image attribution: Zetsche, Bernd, et ...in AT-rich genomes. Additionally, whereas Cas9 generates blunt ends 3 nt upstream of the PAM site, Cpf1...certain tissues. However, these vectors have a low genetic capacity of ~4.5 kb. At 4.1 kb, SpCas9 barely fits...27272384 Find plasmids from this publication at Addgene. 2. Kleinstiver, Benjamin P., et al. "Genome-wide...

...Click on the gene names in the figure below to find plasmids containing that particular gene. ...Additional Resources on the Addgene Blog Learn More about Plasmids for Endogenous Gene Tagging in Human Cells...FP properties. It is important to look out for general problems associated with fluorescent imaging such...constructs you create in your own work available to the Addgene community. Download our Markers of Subcellular ...PMCID: PMC4777285. Further Reading Microscopy U. Generfal Considerations when Using Fluorescent Proteins...Collection Get Tips for Using FRET Resources on Addgene.org Browse the Allen Institute for Cell Science ...

...Truncated gRNAs for Regulating Gene Expression and CRISPR 101: Epigenetics and Editing the Epigenome (plus...Joining and PITCHing MMEJ as an Alternative Route for Gene Editing Day 9: Precision editing: Single Base Editing...Since the start of the CRISPR revolution, Addgene has distributed over 100,000 CRISPR plasmids. But that...flame for its adenine base editing capabilities. Epigenetics: CRISPR has made it possible to design programmable... blue flame plasmids covering three different epigenetic effectors. The diversity of tools is especially...plus bonus podcast with Addgene Advisory Board member Alex Chavez) Day 11: Pooled libraries: Genome-Wide...

...used to improve CRISPR-based activation of target genes, but we’ll save that application for another day...protein-tagging system for signal amplification in gene expression and fluorescence imaging.” Cell 159(3...mitochondria without organelle toxicity. First generation (v1) SunTag is expressed at very low levels due...protein-by-protein basis. However, since many of Addgene’s SunTag plasmids have coveted “blue flames,” it...PMCID: PMC4252608. Additional Resources on the Addgene Blog Sign Up to Receive the Fluorescent Protein...about Small Molecule Fluorphores Resources on Addgene.org Find Fluorescent Protein Empy Backbones Visit...

...biosensors To focus on a particular gene or protein, check out our gene-specific biosensors. These constructs...Visualization Resources on Addgene.org Find Biosensor Plasmids Find Optogenetics Plasmids Visit the Fluorescent... to the molecule it detects. This change then generates a reporter signal. Reporter signals may be electrochemical... OR enzyme activity. Small molecule biosensors Addgene has many sensors that measure the concentration..., and voltage across membranes. These sensors generally follow the same pattern described above - a sensing...fluorescent molecule   Additional Resources on the Addgene Blog Check out the Rosella Autophagy Sensor Read...

...and caveats of Cre usage: Vagaries of conditional gene targeting. Schmidt-Supprian M, Rajewsky K. Nat Immunol...activity regulation. Heidmann D, Lehner CF. Dev Genes Evol. 2001 Sep;211(8-9):458-65. doi:10.1007/s004270100167...specific recombination site for each enzyme, were generated to test recombination efficiency. When expressed....1111704108. Epub 2011 Aug 9. PubMed. Find the Plasmids @Addgene: Plasmids for KD, R, B2, and B3 recombinases from...

...attach the gene of interest to either the N-or the C-terminus. Even if a researcher’s gene of interest...researchers as we could, and Addgene is a wonderful vehicle for us to do that.” At Addgene, we are equally excited...is available in an easily searchable format on Addgene’s Michael Davidson Fluorescent Protein Collection...collection with over 300 backbones alone,” said Addgene’s Lianna Swanson, who has been working with members...the scientific community," says Joanne Kamens, Addgene's Executive Director, "and to promote the great ...