...for CRISPR/Cas9 editing in C. elegans: Engineering the Caenorhabditis elegans genome using Cas9-triggered...2013, a remarkable nine papers describing CRISPR/Cas9 genome engineering methods for C. elegans were released...Multiple features of C. elegans have made CRISPR/Cas9 so successful in this species, namely, the robust...create a new and efficient selection-based CRISPR/Cas9 method to generate knock-ins on any genetic background...background. Their new method uses a selection cassette containing genes for hygromycin resistance and the...heat-shock inducible Cre to generate a self-excising cassette (SEC). To make it possible fluorescently label...into the genome. Figure 1: Self-excising cassette schematic from Dickinson et al. (2015). Used with...

...Enzymatic domains tethered to dCas9 often interact with DNA independently of dCas9 targeting (Galonska 2018...binding is crucial for dCas9 to interfere with DNMT1 even through replication. The dCas9 also needs to be removeable...has several great options: pLV hUbC-dCas9-T2A-GFP for removable dCas9 expression and pLM-CMV-R-Cre. Selection... Some enzymatic fusions are godsends. Tethering dCas9 to the histone acetyltransferase p300 or the DNA...absent before. But other enzymatic fusions, such as dCas9-TET, can, like the iron ball-and-chain that they...-based epigenetic approach (spoiler alert: it’s dCas9… with no modifications). Ready to get started?   ...dioxygenase  When I started to work on building a CRISPR/dCas9-based tool for DNA demethylation in 2015, none existed...

...PubMed Central PMCID: PMC5947990. 3. Jarvik JW, Telmer CA. 1998. Epitope tagging. Annu Rev Genet 32: 601-618... Figure 2: Cas9 RNP complexed with 2-part cr:tracrRNA tags genes more effectively than Cas9 complexed ...Enhanced CRISPR/Cas9-mediated precise genome editing by improved design and delivery of gRNA, Cas9 nuclease,...target site. With the widespread adoption of CRISPR/Cas9 technology and rapid improvements in the field, ...set out to develop a hassle-free, highly efficient Cas9 protein-based method for epitope tagging in mammalian...genes at a time using Custom Synthetic cr:tracrRNA/Cas9 Ribonucleoprotein (csRNP) delivery Epitope tagging...delivery of three reagents to cells: a guide RNA, Cas9 protein, and a donor DNA that contains the tag sequence...

...other Cas proteins Read all of our CRISPR blog posts Read about GeoCas9, another thermostable Cas9 Resources...indicates that like GeoCas9 and ThermoCas9, IgnaviCas9 is a type II-C Cas9. IgnaviCas9 is most similar to...thermostable Cas9 enzymes have previously been characterized: GeoCas9 from the Doudna lab and ThermoCas9 from ...Find IgnaviCas9 here! What can you do with IgnaviCas9? Why would a thermostable Cas9 be useful? It can enable...launched in the Quake Lab. Identifying IgnaviCas9, a hyperthermostable Cas9 This year it was wonderful to learn... Ignavibacteriae phylum genome. IgnaviCas9 is a hyperthermostable Cas9 CRISPR nuclease that is active ...assembled genomes, but also led to the discovery of IgnaviCas9, a new CRISPR enzyme from a sample from Mound...

... to CRISPR/Cas9 Technology? Learn More: Addgene's CRISPR/Cas9 Guide History of CRISPR Cas - A tale of... use CRISPR/Cas9 to generate mice with genes knocked out in the insulin signaling cascades in only 4 months...awards that will be bestowed upon discovers of CRISPR/Cas9 genome engineering, Dr. Jennifer Doudna and Dr. ...offered the great analogy of comparing the CRISPR/Cas9 system to Norton Antivirus. Antivirus programs work... crispr/cas9...

...advanced genome editing machinery such as CRISPR-Cas. Figure 1: Schematic of targeted gene knock-out...explorations of the use of AdV for in vivo CRISPR-Cas delivery utilized the Non Homologous End Joining ..., Cowan CA, Rader DJ, Musunuru K (2014) Permanent Alteration of PCSK9 With In Vivo CRISPR-Cas9 Genome ...capacity is large enough to carry the Cas9 gene and a gRNA expression cassette in one single viral particle. ...DNA Adenoviral CRISPR/Cas9 genome editing tools can also be used to deliver Cas9 and DNA templates in ... CRISPR/Cas9 research! To find more information about the adenoviral delivery of CRISPRS/Cas9 using the... out the plasmids at Addgene: pAdSh.PGK.Cas9 (expresses S. pyogenes Cas9 from the PGK promoter) and U6...

...versions of Cas9, nuclease and nickase, but we are now adopting dCas9, dCas9-VP64, and dCas9-KRAB in our...They went on to show that their CRISPR/Cas9-nuclease and CRISPR/Cas9-nickase vectors work successfully to...all-in-one vector construction system for CRISPR/Cas9-mediated multiplex genome engineering is now available...one of the most advantageous properties of CRISPR/Cas9 compared to ZFNs and TALENs,” said Tetsushi Sakuma...offers ease and efficiency for all-in-one CRISPR/Cas9 vectors expressing up to seven guide RNAs (gRNAs...Figure 1: Schematic overview of the all-in-one CRISPR/Cas9 vector construction system for multiplex genome ...Golden Gate cloning method to ligate expression cassettes of the gRNAs into a single vector in tandem. They...

... screened 11 other Cas9 orthologs to identify a more efficient, well tolerated, Cas enzyme. They identified...binding the DNA or extending the transcript. Different Cas orthologues and subtypes function somewhat differently...from the Lu lab found early success using CRISPR-Cas to target subsets of bacteria in population based...that leverage the remarkable properties of CRISPR-Cas systems is base editing. This process is generally...Fischetti VA, Marraffini LA (2014) Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials...bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Research 41:7429–7437 . https... Streptomyces Species Using an Engineered CRISPR/Cas System. ACS Synth Biol 4:723–728 . https://doi.org...

... the Addgene Podcast.     Additional Resources on the Addgene Blog Listen to Our Podcast Interview with...In the third episode of our Hot Plasmids podcast series, you'll learn about optogenetics tools for controlling...

...Listen to the Fire-Cas9 Podcast Segment Jerry Crabtree’s lab has developed a new CRISPR/Cas9-based system to...epigenetic modulation. While catalytically inactive cas9 (e.g. dCas9) has been fused to a variety of protein modules...Inducible Recruitment for Epigenome editing by Cas9 (FIRE–Cas9), combines these approaches to enable rapid...here. No time to read? Listen to our Hot Plasmids Podcast! TRIM-Away: Targeted endogenous protein degradation...contributed by Mary Gearing Listen to the TRIM-Away podcast segment CRISPR and RNAi have helped researchers...Eric J. Perkins Listen to Endosomal pH Sensors podcast segment The first Addgene plasmid deposit from ...Communications. 2017. PubMed PMID: 28924139. FIRE-Cas9 for inducible interrogation of the epigenome Article...