...Resources on the Addgene Blog: Learn about CRISPR RNA Editing with REPAIR Get tips for using Cas9 Transcriptional...than other Cas13s and a whopping 33% smaller than Cas9! Their small size makes then easy to package in ...significant off-targets, CasRx displayed zero. Unlike Cas9, for which efficiency varies widely across guide...In this case, Konermann et al. targeted MAPT, the gene encoding dementia-associated tau, delivering dCasRx...possibilities. To identify Cas13d, both groups used very general bioinformatic screens that looked for a CRISPR ...regulating methods: small hairpin RNA interference, dCas9-mediated transcriptional inhibition (CRISPRi), and...scientific sharing will continue to grow, and we at Addgene appreciate our depositors for making their tools...

...Delivery Resistance Co-expressed Cas9 Depositing lab Cas9 species = S. pyogenes (PAM = NGG) 46759 pT7-gRNA ...none, need Cas9 plasmid Chen and Wente 42250 DR274 T7 BsaI In vitro transcription none, need Cas9 plasmid...at a much lower efficiency than NHEJ. ID Plasmid Gene/Insert Vector Type Promoter PI Publication Empty...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...out our Suggest a Plasmid form or e-mail help@addgene.org to help us improve this resource!...

...Energy’s Joint Genome Institute for uncharacterized genes that were proximal to a CRISPR array and the universal...CRISPR-Cas systems containing cas1, cas2, cas4, and a new gene cas14. cas14 encodes for a small Cas protein (40...-Cas systems. There are 24 variants of the cas14 gene that cluster into 3 subgroups (cas14a-c). All these...primitive version of the larger and more complex Cas9 and Cas12 proteins. Cas14 cleaves ssDNA But what...potential of Cas14, the Doudna lab cloned the cas14 gene together with the adjacent CRISPR array and intergenic... and cleave a targeted sequence of ssDNA. Unlike Cas9, Cas14 does not require a PAM sequence. In addition...presence of infectious organisms (ex: SARS-CoV-2) and genetic mutations. By providing a gRNA specific of a viral...

...among other things) gene delivery, generating pluripotent stem cells, and developing gene therapies for monogenic...Cre-dependent gene expression, gene knockout, and selection marker removal. CRISPR technology pdCas9-humanized...and lentiviral vectors that are used for gene regulation, gene of interest expression, knockout, and screening...codon-optimized Cas9 under the control of Murine Stem Cell retrovirus promoter for mammalian gene knockdown....Expressing your gene of interest pLenti-puro (Plasmid #39481) You can clone your gene of interest into...inducible gene overexpression, knockdown (shRNA, miR-30-shRNA), mutation/editing (CRISPR-Cas9) or deletion...This is a 3rd generation lentiviral backbone from the Zhang lab expressing S. pyogenes Cas9 and a gRNA cassette...

... In Resistance Co-expressed Cas9 Depositing lab Cas9 species = S. pyogenes (PAM = NGG) 49014 pRPR1_gRNA...dead dCas9, or dCas9 fused to a transcriptional repressor peptide like KRAB, can knock down gene expression...Plasmid Gene/Insert Promoter Selectable Marker PI Publication Base Edit Catalytically dead dCas9 fused ...Description Gene/Insert Promoter Selectable Marker PI Publication Activate Catalytically dead dCas9 fused to...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 Selectable... plasmid to target the dCas9-repressor to your specific locus. ID Plasmid Gene/Insert Promoter Selectable...

...using CRISPR/Cas9 to edit a gene, the expression of Cas9 is no longer necessary. However, Cas9 can be toxic...Gag, Pol, Rev, and Tat genes. Specifically for our knowledge here, the Pol gene encodes three separate...Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy...insertional mutagenesis. This integration could lead to an undesirable interruption of a critical gene. Because...Insertional mutagenesis is especially concerning in clinical settings, as evidenced by retroviral gene therapy...like creating stable cell lines, delivering CRISPR/Cas9 to cells for genome editing, and introducing inducible...transcriptase), and IN (integrase). These viral packaging genes are required for the production of lentiviral particles...

... available with multiple Cas9 variants, including high fidelity Cas9s, Cas9s with various PAM requirements...dual nickases to generate a knockout or gene edit; or using Cas9 to generate large genomic deletions or ...strand SpCas9-HF1 - disrupt Cas9’s interactions with DNA phosphate backbone HypaCas9 - increase Cas9 proofreading...Plasmids: Purify Cas9 Alternatives for CRISPR Genome Engineering S. pyogenes Cas9 (SpCas9) is the most commonly...new tools. Figure 3: Overview of Cas9n Nickase and dead Cas9 The two Cas9 nuclease domains, RuvC and HNH...enhanced specificity by engineering Cas9 into high fidelity Cas9s (hfCas9) . There is currently no defined...application. Cas9 Orthologs While SpCas9 is the most commonly used Cas enzyme, additional Cas9 orthologs ...

...technology of the year in our collection. CRISPR/Cas9 - At Addgene, 2013 will be known as the "Year of the CRISPR...frequently requested. Ubiquitin - One particular gene we saw increased interest in from 2008 to 2009 was...expression and pLVTHM, a lentiviral plasmid for mammalian gene silencing. TALENs - Genome engineering methods improved.... So far, we have distributed over 10,000 CRISPR/Cas9 plasmids to more than 3,000 labs around the world...Addgene was founded 10 years ago today. In that time, Addgene has shipped over 350,000 individual plasmids...biosensors, optogenetics, and other imaging tools. The GCaMP5 and GCaMP6 series of genetically encoded calcium...Top 10 plasmid technologies distributed through Addgene over our first 10 years. 10 most distributed plasmid...

...Protocol to generate stable cell lines expressing a gene of interest from an integrated lentiviral vector...This protocol can be used to generate stable cell lines expressing a gene of interest from an integrated...A549 cells stably expressing Cas9. A549 cells were transduced with lentiCas9-Blast and then selected with... Protocols Generating Stable Cell Lines with Lentivirus Generating Stable Cell Lines with Lentivirus... may have 1 copy of the transgene, while others have >1 copy of the transgene in their genome. This can...high levels of the transgene may have reduced growth rates, especially if that transgene is toxic. Eventually...low-level transgene expressors may take over the culture. To overcome this, consider generating monoclonal...

...listed below. When targeting other gene loci, you will prepare the gene-specific CRISPR and donor vectors...pX330S-2-PITCh - expresses Cas9 & PITCh-gRNA pX330A-FBL/PITCh - expresses Cas9, PITCh-gRNA, & FBL-specific...Doyon lab, eSpCas9(1.1)_No_FLAG_AAVS1_T2 (Addgene #79888) , which expresses an untagged Cas9 AND a gRNA...Drosophila cells that uses PCR to generate both an expression cassette for the Cas9-programming sgRNA and HR donors...multiple genes. Plasmids can be found associated with the following article: Paix, et al. Genetics 2014 Allen...CRISPR plasmids for endogenous tagging of your gene of interest. CRISPR...repair template design. How to use CRISPR to tag your gene of interest Mendenhall and Myers Tagging System ...

... RNA delivery of Cas9 and gRNA In vitro transcription reactions generate mature Cas9 mRNA and gRNA, which...used to generate transgenic embryos Cas9-gRNA ribonucleoprotein (RNP) complexes Purified Cas9 protein ...knockin. Select gene and genetic element to be manipulated In order to manipulate a given gene using CRISPR...mutant allele of a gene ( point mutant )? Increase or decrease expression of a target gene? Once you have ...disrupt gene function in a particular cell type or organism without a specific preferred mutation Cas9 (or...point mutation or inserting a tag Cas9 (or Cas9 nickase); Base editor Single (or dual) gRNA targeting the...concern, Cas9-gRNA ribonucleoprotein (RNP) complexes are advantageous due to the transient Cas9 expression...

...expression of your gene of interest.  SapTrap Tool Kit for CRISPR Cas9 edits At Addgene, we have the entire...high-throughput CRISPR/Cas9 gene modification in Caenorhabditis elegans." Genetics 202.4 (2016): 1277-1288...toolkits available at Addgene.  C. elegans open resources The C. elegans Neuronal Gene Expression Map & Network...Additional resources on the Addgene blog Learn more about SapTrap Read about CRISPR/Cas9 in C. elegans Browse... The CeNGEN goal is to establish a comprehensive gene expression atlas of every C.elegans  neuron. Scientists... C. elegans embryo when all essential embryonic genes are individually depleted via RNA interference (...based bipartite expression system that can regulate gene expression in C.elegans. In this system a promoter...

...numerous applications, including transgenesis, gene-trap screens, and gene editing. How does the piggyBac...levels of your gene of interest, as needed. Controlling the expression levels of your gene may be particularly...Transposagen.   How does piggyBac™ enhance gene editing? Gene editing can now be accomplished in a more...(CRISPR/Cas9, TALENs, and ZFNs). However, several challenges still remain, such as making gene edits completely... How does the piggyBac™ gene editing system compare with other methods of gene editing? There are primarily... a variety of gene editing technologies including Zinc Fingers, TALENs, and CRISPR-Cas9 nucleases. Mathew...Decoding the Epigenetic Heterogeneity of Human Pluripotent Stem Cells with Seamless Gene Editing.,” Methods...

...Mammalian ID Plasmid Gene/Insert Promoter PI Publication Drosophila ID Plasmid Gene/Insert Promoter PI ...catalytically inactive Cas9 (dCas9) is fused to FokI nuclease. When FokI dimerizes, it generates a double-strand...PI Publication Yeast ID Plasmid Gene/Insert Promoter PI Publication Do you have suggestions for other plasmids... CRISPR dCas9-FokI CRISPR: dCas9-FokI...with dCas9 fused to FokI for creating double-strand breaks (DSBs). CRISPR...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...

... Strong expression: A CMV promoter drives robust gene expression in mammalian cells. Flexible tagging:...tag. Selectable and stable: A puromycin resistance gene makes stable cell line creation a breeze. Modular...Bentley For RNA-guided genome editing, think beyond Cas9 with STITCHR: a combined CRISPR / retrotransposon.... Figure 4: STITCHR consists of a nicking Cas9 fused to a shortened R2Tocc transposon protein, ...for scarless insertion of the payload (teal). The Cas9 gRNA targeting guide and the transposon RNA homology...scientists fused the retrotransposon protein R2Tocc to a Cas9 nickase to improve target recruitment and nicking...labeling By Alyssa Shepard To fully understand how genes are regulated, it's important to know what proteins...

...Plasmid Gene/Insert Selectable Marker PI Publication Purify A catalytically inactive Cas9 (dCas9) can be...(HDR). ID Plasmid Gene/Insert Promoter Selectable Marker Publication Prime Edit Cas9 H840A nickase fused... to immunoprecipitate FLAG-tagged Cas9. In the CAPTURE system, Cas9 is tagged with a biotin acceptor site...Plasmid Gene/Insert Promoter Selectable Marker PI Publication Visualize A catalytically inactive Cas9 (dCas9...dead dCas9, or dCas9 fused to a transcriptional repressor peptide like KRAB, can knock down gene expression...Plasmid Gene/Insert Promoter Selectable Marker PI Publication Activate Catalytically dead dCas9 fused to...transcription of a specific gene. Design your gRNA sequence to direct the dCas9-activator to promoter or ...

...Truncated gRNAs for Regulating Gene Expression and CRISPR 101: Epigenetics and Editing the Epigenome (plus...and CRISPR Antimicrobials Day 7: Multiplexing and Cas9 variants: CRISPR 101: Multiplex Expression of gRNAs...Joining and PITCHing MMEJ as an Alternative Route for Gene Editing Day 9: Precision editing: Single Base Editing...Genome-Wide Screening Using CRISPR/Cas9 and Plasmids 101: NGS Quality Control for Pooled Libraries Day 12: New... CRISPR Targeting Specificity with eSpCas9, SpCas9-HF1, & HypaCas9 Day 10: Transcriptional regulation:...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...

...70% of human genes have a counterpart in zebrafish. Understanding the function of these genes in fish will...unknown functions for genes, and lays the groundwork for understanding the gene networks that regulate...zebrafish-specific gene expression databases that provide information about when and where genes are active in...TALENs, and more recently CRISPR/Cas9. Another popular targeted gene deactivation method in zebrafish ...investigations. Genetic resources for zebrafish A common approach to unravel the gene networks that drive...model organism is its amenability to genetic manipulation. Tools for gene manipulation together with information...Additional mutagenesis projects have since been initiated and today thousands of mutations in genes affecting...

...lab utilizes a CRISPR/Cas9 based approach to knockout, repress, and activate gene targets. To accomplish...fusion for gene activation CRISPR/Cas9 Plasmid Variants Figure 1: Schematic of the CRISPR Collection. Plasmids... plasmids dCas9-KRAB-MeCP2 fusion for gene and non-coding enhancer sequence repression dCas9-VPR fusion...CRISPR Collection The Brzezinski lab investigates gene regulation in the context of the developing mouse...taken advantage of the Zhang lab all-in-one (guide/Cas9/reporter) pX458 plasmid family. They have made several...pX458 plasmid to expand its usefulness for the lab's gene regulation and developmental studies. Key plasmid...fate specification, yet differs from deleting both genes . Development. doi: 10.1242/dev.190272. PMID: 32541005...

...found in the same gene. If we only have small, directed editing abilities, any gene editing treatment ...functional copy of the entire gene, allowing a one-size-fits-all approach to gene replacement therapy for most...programmable gene insertion with PASTE. The PASTEsystem involves insertion of landing sites via Cas9-directed...insertions, leaving a several kilobase-sized gap in the gene editing field. The AbuGoot lab has now developed...to work. (It was also found to be independent of gene repair.) This lowers the chance of the insert being...activity. They now had a system that could integrate genes up to 36 kb in length, with an integration rate ...bylanding site recognition and integration of cargo via Cas9-directed integrases.   Use PASTE can be delivered...