... sites for S. pyogenes Cas9, S. aureus Cas9, S. thermophilus Cas9, N. meningitidis Cas9, or Cas12a from...vectors using ssDNA oligos p201G Cas9 ; p201B Cas9 ; p201H Cas9 ; p201N Cas9 ; pUC gRNA Shuttle PDF, 500 KB...CRISPR RNA array: Cas9 (pX260) or Cas9 D10A (pX334) ; tracrRNA: Cas9 (pX330) or Cas9 D10A (pX335) PDF,... window) CRISPR/Cas9 reagent generation; gRNA design; HDR design; Deep sequencing Cas9: pMLM3613 dsDNA...CRISPR/Cas9 Knockout (MAGeCK). This tool can identify positively or negatively selected sgRNAs, genes, or...118 KB Church gRNA design and cloning for Cas9 orthologs Cas9 plasmids PDF, 107 KB Chen and Wente Zebrafish...vitro transcription, injection gRNA core ; Cas9 ; optimized Cas9 PDF, 66.8 KB Fujii gRNA design and cloning...

...Return to top Cas9 SAM transcriptional activation plasmids and screening library CRISPR/Cas9 Synergistic... of endogenous genes. It consists of three components: A nucleolytically inactive Cas9-VP64 fusion A sgRNA...of cells and nuclei. Return to top AAV - Cas9 mouse CRISPR-Cas9 is a versatile genome editing technology...constitutively expressing Cas9 knockin mice (Platt et al ., Cell 2014). In these mice the CRISPR-Cas9 system can be...constitutively Cas9-expressing mouse. Described here are AAV vectors that can be combined with Cas9 in a wide...are below. The Cas9 knockin mouse can be purchased from Jackson Labs: Cre-dependent Cas9 mouse Constitutively...cloning information: CRISPR-Cas9 mouse toolbox protocol 637.8 KB CRISPR-Cas9 Cre expression vectors for...

... 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 ...

... 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...

... 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...

...Co-expressed Cas9 Depositing lab Cas9 species = S. pyogenes (PAM = NGG) 47549 pDD162 (Peft-3::Cas9 + Empty ...need Cas9 plasmid Joung 46170 PU6::klp-12_sgRNA cU6 PCR (see paper) Transfection none, need Cas9 plasmid... none, need Cas9 plasmid Boxem 47941 pMB60 T7 BsaI In vitro transcription none, need Cas9 plasmid Boxem...NHEJ. 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...or regulatory regions of your gene of interest. If the plasmid that you choose does not also express a ...

...Delivery Resistance Co-expressed Cas9 Depositing lab Cas9 species = S. pyogenes (PAM = NGG) 49410 pCFD3-dU6...repair (HDR). ID Plasmid Gene/Insert Promoter PI Publication Prime Edit Cas9 H840A nickase fused to a ...transcription Virmilion none, need Cas9 plasmid Bullock and Port 49330 pAc-sgRNA-Cas9 dU6 BspQI Transfection Puromycin..., need Cas9 plasmid Bullock and Port 51026 U6-BbsI-crRNA dU6 BbsI Transfection none, need Cas9 plasmid...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...

...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...

...High fidelity Cas9s: Three high fidelity Cas9 variants , SpCas9-HF, eSpCas9, and HypaCas9 display very ...III and Cas9 enzymes, which together separate the individual crRNAs. (3) The tracrRNA and Cas9 nuclease... anti-CRISPR genes in bacteriophages infecting Pseudomonas aeruginosa . Anti-CRISPR genes employ varied...Staphylococcus aureus (SaCas9) is smaller than SpCas9 and more easily packaged in AAV. Cas9 orthologs also have...especially amenable to genetic screens. Pooled gRNA libraries can be used to identify genes that are important...beyond SpCas9, moving us closer to being able to target every locus in every genome. Multiple Cas9 orthologs...capability of Cas9 to make other types of modifications at specific loci. Activate or Repress : dCas9 fused to...

...backbone. Zhang Cas9 and Accessories for Activating Gene Expression Catalytically-dead Cas9 (dCas9) can be fused...mouse genes along with 1000 non-targeting controls Puromycin Doench , Root Cas9 Lentivirus Cas9 viruses... 52962 lentiCas9-Blast Sp Cas9 Cut Blasticidin Expresses human codon-optimized S. pyogenes Cas9 protein...page . Cas9 for Editing Genomic Sequences To perform genome edits, Cas9 nuclease is used to generate double-stranded...backbone contains SpCas9. SpCas9 and 76,441 unique sgRNAs targeting 19,114 human genes along with 1000 ...Lentivirus Pooled CRISPR Libraries Cas9 Pooled Barcoding Libraries Control Addgene's lentiviruses are made from...backbone does not contain SpCas9. 76,441 unique sgRNAs targeting 19,114 human genes along with 1000 non-targeting...

...can knock down genes by feeding worms bacteria expressing dsRNA or use CRISPR/Cas9 gene editing to easily...briggsae , etc.) gene or sequence: ID Plasmid Gene/Insert PI Don’t see your gene listed? Know of some great... via a CRISPR-Cas9 system. Cloning-free CRISPR for C. elegans , which uses purified Cas9, such as that...Tagging of Endogenous Genes with SapTrap Even more elegant: Single injection CRISPR/Cas9 in C. elegans External...Custom Genetic Modifications with CRISPR/Cas9 in Caenorhabditis elegans. Mos1 Based Transgenesis miniMos... used in worms, search Addgene's entire collection. ID Plasmid Description Gene/Insert PI Plasmids Expressing...which diminishes epigenetic silencing in the germline. A "FLP-Out" system for controlled gene expression in...

...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...

...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...

... 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...

...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...

...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...

...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 ...

...zebrafish gene of interest? Search Addgene's collection for plasmids that contain a zebrafish gene or sequence...Lab CRISPR/Cas9-based conditional mutagenesis in zebrafish - Wenbiao Chen Lab A CRISPR/Cas9 Vector System... zinc finger nucleases, TALENS, and CRISPR/Cas9-based gene editing make the zebrafish a stalwart research...zebrafish-specific gene expression database that provides information about when genes are active in developing...developing embryos. zfRegeneration - A database for gene expression profiling during regeneration. Seurat - R ...-type specific conditional gene inactivation - Jeffrey Essner Lab GeneWeld Vectors for Targeted Integration...modified CRISPR-Cas9 system - Shuo Lin Lab Re-purposed Ac/Ds transgenic system enabling CRISPR/dCas9-interference...

...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!...