...isoform-specific gene expression. Gene isoforms refer to mRNAs generated from the same gene but with different...variant of Cas9 (dCas9) and engineered combinations of transcriptional activators to induce gene transcription...isoform-specific gene regulation — at least, more straightforward than generating cDNA for each gene isoform individually...first ask yourself whether gene overexpression is necessary (instead of gene knockout or knockdown, for...with neuron-specific expression of dCas9-VPR (Figure 2), a popular Cas9 activator developed by the Church... neuron-optimized CRISPR/dCas9 activation system for robust and specific gene regulation." eNeuro 6.1 ...transcriptional activation of multiple genes in the brain using CRISPR–dCas9-activator transgenic mice." Nature...

... - this time using the Staphylococcus aureus Cas9 (SaCas9). This AAV-based genome editing system is regulated...the Cas12b plasmids  Plant base editing with a Cas9-adenosine deaminase fusion Caixia Gao’s lab has ...tRNA adenosine deaminase that is fused to a nicking Cas9. This base editor can be used for A•T to G•C edits... a few of the new CRISPR plasmids available at Addgene. We will still periodically focus on specific CRISPR... in Molecular Neuroscience Find the AAV-based SaCas9 genome editing plasmids String assembly gRNA ...is a single step gRNA multiplexing system that generates vectors containing high numbers of gRNAs in one...a new CRISPR tool you’ve recently deposited to Addgene and you’d like it to be included in the next What...

... plasmid contains Cas9 and if so, which function of Cas9. We have plasmids with Cas9 that can cut, activate... Co-expressed Cas9 Cas9 System Selection PI pCRISPR 42875 Bacteria BsaI none S. pyogenes Chloramphenicol...meningitidis Joung Cas9 sgRNA vector 68463 Mammalian U6 none S. pyogenes Zhang Cas9/pTREX-n 68708 Other/...independent U6 promoters and Cas9 from CBh promoter. Ventura A CRISPR/Cas9 toolkit for multiplex genome...polycistronic glycine tRNA-gRNA genes (PTGs) and then PTG cassettes are assembled into a Cas9 expressing vector. Yang...none S. pyogenes Qi pDD162 (Peft-3::Cas9 + Empty sgRNA) 47549 C. elegans yes, cut S. pyogenes Goldstein...cut S. pyogenes mCherry Kuhn pU6-(BbsI)_CBh-Cas9-T2A-BFP 64323 Mammalian BbsI yes, cut S. pyogenes BFP Kuhn...

...the Cas9 nickase doesn't have the ability to induce double-stranded breaks like the wildtype Cas9 does... engineering experiments, consider that wildtype Cas9 with optimized chimeric gRNA has high efficiency...effect). The double nickase system is based on the Cas9 D10A nickase described in Figure 4 of the Cong, ...can express two different chimeric gRNAs with the Cas9 nickase which will together introduce cleavage of...thorough step-wise instructions on designing and using Cas9-based genome engineering tools to perform different...PAMs in the region, will they also be targeted? The Cas9 cuts 3-4bp upstream of the PAM sequence. There can... can be some off-target DSBs using wildtype Cas9. The degree of off-target effects depends on a number...

...domain due to a gene that resembles cas10. All type IV subtypes are missing adaptation genes, Cas1 and Cas2...–C, with the beloved SpCas9 (from Streptococcus pyogenes) belonging in II-A. Cas9 was the first to be ...sequence similarity, phylogenetic analysis, neighborhood analysis of surrounding genes, and experimental ...first time. The discovery and application of CRISPR-Cas9 kicked off over a decade of frenzied interest in...straightforward as it seems. There is not a single gene that is shared across all CRISPR types; the fast...while Class 2 use single protein Cas effectors, like Cas9. The Cascade complex doesn’t usually carry out the...together because they contain a distinct Cas7-type gene. The exact mechanism of type IV systems is not known...

...hammer: Gene knockout by NHEJ Gene knockout with CRISPR technology is usually accomplished by Cas9-mediated...technologies – a nuclease-dead Cas9 (dCas9) is directed near the promoter of a target gene. Here, the target window...you are trying to do: gene knockout, a specific base edit, or modulation of gene expression. Location ...multiple sequences targeting a gene to score in order to call that gene a hit.  This is an exciting time...Agreement between two large pan-cancer CRISPR-Cas9 gene dependency data sets. Nat Commun 10: . https:/...protein-coding capacity of a locus. When using S. pyogenes Cas9 (SpCas9), potential target sites are both [5’-20nt-NGG...This means that, for gene editing, there are usually few potential gRNAs. While SpCas9, with a PAM preference...

...editing technique that relies on a fusion enzyme of Cas9 nickase + reverse transcriptase and a prime editing...., 2019), disrupting its canonical structure and Cas9 binding (Nishimasu et al., 2014). Figure...pegRNAs that use other bases in this location bind to Cas9 with canonical sgRNA structure (right). Created ...along with your intended edit. This prevents the Cas9 nickase from re-binding and nicking the newly synthesized...Zhang, F., & Nureki, O. (2014). Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA. Cell, ...and goal of the experiment. Now, onto the tips! General principles pegRNA design If you’re just starting...programs to help you design pegRNAs. Here on the Addgene blog, we featured a guest blog by the developer...

...the dramatic spread of the Cas9 technology: "The rapid adoption of the Cas9 technology was also greatly...program are here. This was a great opportunity for Addgene to present our own data on plasmid deposits and...and distirbution for these fast moving fields.  Addgene is a global nonprofit plasmid repository. Over ...Over 2,000 labs have deposited plasmids to Addgene and we distribute over 130,000 plasmids in 2014. Thus,...combination of open-source distributors such as Addgene, as well as a number of online user forums such...Meeting Eric presented a poster with data comparing Addgene global deposit and distribution trends for Zinc...

...CRISPR/Cas9 assembly for fast generation of multiple Arabidopsis mutants - Niko Geldner. CRISPR-Cas9 vector... an intronized Cas9 series of vectors and DNA modules for assembly of CRISPR/Cas9 constructs - Sylvestre... a new window) - Tools for gene and protein expression and mapping of gene expression in plants. Figshare...MoClo-compatible zCas9i Cloning Kit includes a high efficiency intron-optimized SpCas9-coding gene, zCas9i, and an...from genome engineering and gene regulation to fluorescent proteins. Addgene’s collection includes plasmids...plasmids containing promoters that drive general and species-specific gene expression (see the column ‘promoter...vectors: A modular plasmid assembly kit for multigene expression, gene silencing, and silencing rescue in plants...

...codon-optimized Cas9 constructs 44249 pdCas9-bacteria A catalytically inactive bacterial Cas9 expression plasmid...fold repress on targeted genes. 2. Two-plasmid CRISPRi system for mammalian gene knockdown The first plasmid...sgRNA targeting endogenous CD71 gene 46919 pMLS-SV40-EGFP Target EGFP gene that is stably integrated into...plasmid ( pdCas9_humanized ) contains a human codon optimized dcas9 gene under the control of Murine Stem...endogenous CXCR4 gene 46920 pTDH3-dCas9 Yeast CEN/ARS vector (Leu2) that contains dCas9 fused to NLS controlled...machineries, requiring only a nuclease-deficient Cas9 (dCas9) protein and a customized s ingle g uide RNA ...basepair complementary region to any gene of interest. Co-expression of dCas9 and sgRNA can efficiently block...

...are created by fusing Cas9 nickase or catalytically inactive “dead” Cas9 (dCas9) to a cytidine deaminase...editors are available with multiple Cas9 variants including high fidelity Cas9’s. Further advancements have ... ID Plasmid Gene/Insert Promoter Selectable Marker PI Publication Bacteria ID Plasmid Gene/Insert Promoter...Plant ID Plasmid Gene/Insert Promoter Selectable Marker PI Publication Yeast ID Plasmid Gene/Insert Promoter...AID-mediated mutagenesis (TAM) from the Chang lab and CRISPR-X from the Bassik lab , in which Cas9 is fused...base editors, the evolved TadA domain is fused to a Cas9 protein to create the adenine base editor. Both ...Selectable Marker PI Publication Zebrafish ID Plasmid Gene/Insert Promoter Selectable Marker PI Publication...

... on therapeutic strategies that use gene replacement therapy, gene editing, MECP2 reactivation, RNA editing...Novartis and rebranded to Novartis Gene Therapies, and Taysha Gene Therapies, founded on Gray’s work, ...corrected by CRISPR/Cas9 in wild-type human iPSCs. Image modified from Le et al., 2019. Gene editing via ...the CRISPR/Cas9 system to both precisely insert the MECP2-R270X mutation into the MECP2 gene in wild-type...protein 2 (MECP2) gene, Rett Syndrome is a rare, progressive, neurodevelopmental genetic disorder that affects... CRISPR/Cas9-Mediated MECP2 Modifications in Human-Induced Pluripotent Stem Cells. Front Genet 10: . https...of Rett Syndrome research, the role of the MECP2 gene, and highlights some of the latest breakthrough ...

... In Resistance Co-expressed Cas9 Depositing lab Cas9 species = S. pyogenes (PAM = NGG) 42875 pCRISPR BsaI...repair (HDR). ID Plasmid Gene/Insert Promoter PI Publication Prime Edit Cas9 H840A nickase fused to a ...dead dCas9, or dCas9 fused to a transcriptional repressor peptide like KRAB, can knock down gene expression... ID Plasmid Gene/Insert Promoter PI Publication 64325 3xFLAG-dCas9/p-bacteria 3xFLAG-dCas9 pLtetO-1 Fujii...template. ID Plasmid Gene/Insert Promoter PI Publication Activate Catalytically dead dCas9 fused to a transcriptional...transcription of a specific gene. Design your gRNA sequence to direct the dCas9-activator to promoter or ... plasmid to target the dCas9-activator to your specific locus. ID Plasmid Gene/Insert Promoter PI Publication...

...critical for Cas9 cleavage), thereby rendering the resulting construct resistant to Cas9 induced cleavage... introduced to endogeneous DNA through homology-directed repair (HDR) of a CRISPR/Cas9 induced double-...resistant to Cas9 cleavage. If a plasmid contains the template, site-directed mutagenesis can be used ...footnote; a similar approach can be used to identify Cas9/CRISPR-induced genome modifications. Occasionally... the mutagenesis reaction. Alternative approaches to site directed mutagenesis Point-mutagenesis is fairly...relevant in this age of CRISPR; site-directed mutagenesis generally applies to plasmids, but may also facilitate...modifications.   Site-directed mutagenesis of Plasmids. A) Generation of mutagenized plasmids. PCR primers...

...replace” gene editing method in which a reverse transcriptase (RT) is fused to the C terminus of Cas9 H840A... ID Plasmid Gene/Insert Promoter Selectable Marker PI Publication Bacteria ID Plasmid Gene/Insert Promoter...ID Plasmid Gene/Insert Promoter Selectable Marker PI Publication Drosophila ID Plasmid Gene/Insert Promoter...contains Cas9. ID Plasmid Expression System Promoter Guide RNA Type Cloning Enzyme Co-expressed Cas9 Selection...directs the Cas9 nickase to the target sequence where it nicks the non-target strand and generates a 3’ flap...nuclear localization signals, and two mutations in Cas9 to enhance nuclease activity; can be used in PE2...PE6 - small prime editors with optimized RT and/or Cas9 domains PE7 - addition of RNA-binding exonuclease...

...using bacterial phage vectors with CRISPR/cas9 sensitizing gene cassettes. Thse vectors are engineered to... tetracycline cas9 inducible knock-out system. His lab is also exploring the use of cas9 proteins from...were identified for 61 such diseases. Now single gene alleles have been identified for 4,163, over 50%...Lowe talked about his work on the 500+ human tRNA genes and, until now, we've overlooked regulatory fragments...may be involved in preventing silencing of self-genes. Dr. Mello presented a hypothesis that these pathways...involved in RNA based surveillance of repetitive gene families and provide a way to guide inheritance ... to replace the drug resistantance genes in the bacteria thus making the bacteria sensitive to known antibotics...

...Delivery Resistance Co-expressed Cas9 Depositing lab Cas9 species = S. pyogenes (PAM = NGG) 51306 pUC57-Simple-gRNA...designed for use in Xenopus. Cut Fully functional Cas9 enzymes designed to introduce a double-strand break...surrounding the DSB is introduced along with the Cas9 and gRNA plasmids, the cell may instead repair the...faithfully introduce specific genomic changes. ID Plasmid Gene/Insert Promoter PI Publication Empty gRNA Expression...backbone T7 BsaI In vitro transcription none, need Cas9 plasmid Chen Do you have suggestions for other plasmids...Transcriptional Regulation Activate Interfere Epigenetics RNA Targeting RNA Targeting RNA Editing Other...Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled Libraries gRNAs Empty gRNA Vectors gRNAs...

...possible deletion strategies for genes and non-coding elements. For creating a gene knockout, two sgRNA located...evaluate the RNA, perform RT-qPCR for gene expression of the relevant gene 7,8 . To evaluate the protein, perform...Article Citation Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9. Bauer DE, Canver... CRISPR/Cas9 is an inexpensive, facile, and efficient genome editing tool that allows genetic perturbation...perturbation of genes and genetic elements. Here we present a simple methodology for CRISPR design, cloning,...for efficient loss-of-function studies of genes and genetic elements in mammalian cell lines. Protocol...excerpted from: Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9. Bauer DE, Canver...

...specifity of Staphylococcus aureus Cas9 (SaCas9 KKH variant) Engineered CRISPR-Cas9 nucleases with altered PAM...the Cas9 endonuclease) that cleaves the target DNA. The Joung lab recently described gRNA and Cas9 expression...for the EGFP Reporter Gene Guide RNA expression plasmids for endogenous human genes Guide RNA expression...these vectors for efficiently modifying endogenous genes in zebrafish (Hwang and Fu et al., Nat Biotechnol...expression plasmids for endogenous zebrafish genes... (AsCpf1 and LbCpf1) for use in mammalian cells SpCas9 variants with no detectable off-target effects ...

...engineered Cas9 variant might work for your experiment. PITChing MMEJ as an Alternative Route for Gene Editing...Corn’s lab describe how a better understanding of Cas9 biophysics can help enhance HDR. CRISPR 101: Ribonucleoprotein...tool to help you design, pick, and close guides for Cas9 and Cpf1. This tool supports over 250 genomes, and...template to increase efficiency of insertion and gene replacement. Care to share your favorite CRISPR ...Blugene and I represented Addgene at the recent Keystone meeting on Precision Genome Editing with Programmable...hopes to evaluate genome editing pipelines and generate a common lexicon for genome editing studies. These...desired edit, and I want to make sure that I (and Addgene) don’t forget this in the future. Here’s a list...