...vectors using ssDNA oligos p201G Cas9 ; p201B Cas9 ; p201H Cas9 ; p201N Cas9 ; pUC gRNA Shuttle PDF 488.6 ...CRISPR RNA array: Cas9 (pX260) or Cas9 D10A (pX334) ; tracrRNA: Cas9 (pX330) or Cas9 D10A (pX335) ; PDF...putative target sites for S. pyogenes Cas9, S. thermophilus Cas9, or Cpf from your input sequence or list....2 KB Church gRNA design and cloning for Cas9 orthologs Cas9 plasmids PDF 104.1 KB Chen and Wente Zebrafish...vitro transcription, injection gRNA core ; Cas9 ; optimized Cas9 PDF 66.8 KB Fujii gRNA design and cloning... Injection and selection for Cas9-triggered homologous recombination Cas9-sgRNA target construct ; pMA122...destination: pAC90 PDF 1.0 MB Katic Nematode: Cas9 and gRNA use Cas9 (pIK86) ; gRNA empty backbone (pDR274) ...

...Return to top Cas9 SAM transcriptional activation plasmids and screening library CRISPR/Cas9 Synergistic...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... (20bp). CRISPR-Cas9 Cre expression vectors for genome editing in the brain Using Cas9 mice Platt et al...

... available with multiple Cas9 variants, including high fidelity Cas9s, Cas9s with various PAM requirements...strand SpCas9-HF1 - disrupt Cas9’s interactions with DNA phosphate backbone HypaCas9 - increase Cas9 proofreading...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 ...constructs with Cas9 fused to proteins that modulate HDR or NHEJ components, like miCas9 , Cas9-DN1S , or others...Plasmids: Purify Cas9 Alternatives for CRISPR Genome Engineering S. pyogenes Cas9 (SpCas9) is the most commonly...

...organism without a specific preferred mutation Cas9 (or Cas9 nickase) Single (or dual) gRNA targeting 5′ ...generating a point mutation or inserting a tag Cas9 (or Cas9 nickase); Base editor Single (or dual) gRNA ...concern, Cas9-gRNA ribonucleoprotein (RNP) complexes are advantageous due to the transient Cas9 expression... RNA delivery of Cas9 and gRNA In vitro transcription reactions generate mature Cas9 mRNA and gRNA, which...generate transgenic embryos Cas9-gRNA ribonucleoprotein (RNP) complexes Purified Cas9 protein and in vitro transcribed...and/or Cas9 expression or (2) a lack of efficient target cleavage in cells expressing both Cas9 and gRNA...research. Although we will use the example of CRISPR/Cas9 in mammalian cells, many of these principles apply...

... plasmid contains Cas9 and if so, which function of Cas9. We have plasmids with Cas9 that can cut, activate...Expression System Cloning Enzyme Co-expressed Cas9 Cas9 System Selection PI pCRISPR 42875 Bacteria BsaI...BbsI)_CBh-Cas9-T2A-BFP 64323 Mammalian BbsI yes, cut S. pyogenes BFP Kuhn pU6-(BbsI)_CBh-Cas9-T2A-mCherry...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...cassettes are assembled into a Cas9 expressing vector. Yang A Robust CRISPR/Cas9 System for Convenient, High-Efficiency...accessory pack is available for dCas9 and FokI-dCas9. Yamamoto Multiplex CRISPR/Cas9-based genome engineering...

...links below: ID Plasmid 46294 pBS-Hsp70-Cas9 : A codon-optimized Cas9 nuclease under the control of the Drosophila...Drosophila hsp70 promoter. 45945 pHsp70-Cas9 : The codon-optimized Cas9 nuclease under the control of the Drosophila...Browse CRISPR Cas9 gRNA plasmids for drosophila research from the O'Connor-Giles, Wildonger, and [email protected] We have found that a variety of Cas9-mediated genome modifications can be efficiently...alterations can be generated within a month. phsp70-Cas9 and a single chiRNA plasmid can be coinjected to...imperfectly repaired DSBs. Coinjection of phsp70-Cas9 and plasmids encoding two chiRNAs can induce defined...engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease. Gratz SJ, Cummings AM, Nguyen JN, Hamm...

...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...sgRNA-scaffold SP6 AflII In vitro transcription none, need Cas9 plasmid Sternberg 177783 L4440_BioBrick-sgRNA T7...T7 BbsI, BsaI Ingestion by C. elegans none, need Cas9 plasmid Ristow Do you have suggestions for other...Editing Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled Libraries gRNAs Empty gRNA Vectors...Promoter PI Publication Activate Catalytically dead dCas9 fused to a transcriptional activator peptide can...

...Enzyme(s) Delivery Resistance Co-expressed Cas9 Depositing lab Cas9 species = S. pyogenes (PAM = NGG) 49410...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...Plasmid Gene/Insert Promoter PI Publication Prime Edit Cas9 H840A nickase fused to a reverse transcriptase (...Injection or in vitro transcription Virmilion none, need Cas9 plasmid Bullock and Port 49411 pCFD4-U6:1_U6:3tandemgRNAs...pU6-BbsI-chiRNA dU6 BbsI Transfection none, need Cas9 plasmid O'Connor-Giles, Harrison, Wildonger 49408...Injection or in vitro transcription Virmilion none, need Cas9 plasmid Bullock and Port 49409 pCFD2-dU6:2gRNA dU6...

...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...Staphylococcus aureus (SaCas9) is smaller than SpCas9 and more easily packaged in AAV. Cas9 orthologs also have...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...the crRNA would bind. (5) After the complex binds, Cas9 separates the double stranded DNA target and cleaves...Single multi-domain effector) Type I (Cas3) Type II (Cas9) Type III (Cas10) Type V (Cpf1) Type IV (Csf1) Type...

...Root Cas9 Lentivirus Cas9 viruses can be used to interrogate the genome in a variety of ways. Cas9 proteins... 52962 lentiCas9-Blast Sp Cas9 Cut Blasticidin Expresses human codon-optimized S. pyogenes Cas9 protein...backbone. Zhang Cas9 and Accessories for Activating Gene Expression Catalytically-dead Cas9 (dCas9) can be fused...plasmids resource page . Cas9 for Editing Genomic Sequences To perform genome edits, Cas9 nuclease is used to...Browse In-Stock Lentivirus Pooled CRISPR Libraries Cas9 Pooled Barcoding Libraries Control Addgene's lentiviruses...Resistance Activity PI 61422 dCAS9-VP64_GFP dCAS9 (D10A, H840A) none Expresses dCAS9-VP64 activator with 2A ...-targeting controls. This backbone contains SpCas9. SpCas9 and 76,441 unique sgRNAs targeting 19,114 human...

... via a CRISPR-Cas9 system. Cloning-free CRISPR for C. elegans , which uses purified Cas9, such as that...feeding worms bacteria expressing dsRNA or use CRISPR/Cas9 gene editing to easily engineer specific transgenic...worm CRISPR resources include plasmids expressing Cas9, empty gRNA backbones, and constructs for endogenous...targeting vectors for tag insertion using CRISPR/Cas9 and the selection of C. elegans strains. CRISPR ...that expressed from pHO4d-Cas9 from Michael Nonet. Protocol can be found at the (Link opens in a new window...Recovery of Custom Genetic Modifications with CRISPR/Cas9 in Caenorhabditis elegans. Mos1 Based Transgenesis...Blog Posts An “elegans” Approach to Better CRISPR/Cas9 Editing Efficiency Fluorescent Tagging of Endogenous...

...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 ...base editors, the evolved TadA domain is fused to a Cas9 protein to create the adenine base editor. Both ... lab and CRISPR-X from the Bassik lab , in which Cas9 is fused to activation-induced cytidine deaminase...Editing Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled Libraries gRNAs Empty gRNA Vectors...

... an intronized Cas9 series of vectors and DNA modules for assembly of CRISPR/Cas9 constructs - Sylvestre...CRISPR/Cas9 assembly for fast generation of multiple Arabidopsis mutants - Niko Geldner. CRISPR-Cas9 vector...plasmids expressing different functional variants of Cas9 and empty gRNA backbones. A few examples of useful...truncatula , wheat, and barley - Daniel Voytas. A CRISPR/Cas9 Toolkit for use in a variety of plant species (e.g...or more gRNA expression cassettes - Qi-Jun Chen. Cas9 and gRNA backbone for use in rice. Includes a protocol...species and genotypes - Zhenghe Li. A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex...MoClo-compatible zCas9i Cloning Kit includes a high efficiency intron-optimized SpCas9-coding gene, zCas9i, and an...

...codon-optimized Cas9 constructs 44249 pdCas9-bacteria A catalytically inactive bacterial Cas9 expression plasmid...machineries, requiring only a nuclease-deficient Cas9 (dCas9) protein and a customized s ingle g uide RNA ...Description 44246 pdCas9-humanized A catalytically inactive, human codon-optimized Cas9 expression plasmid...plasmid 44247 pdCas9::BFP-humanized A catalytically inactive, human codon-optimized Cas9-BFP fusion expression... plasmid 44250 pwtCas9-bacteria A wild-type Cas9 expression plasmid for use editing bacterial genomes ...plasmid for use with the bacterial Cas9 constructs 46910 pHR-SFFV-dCas9-BFP Human expression vector containing...CRISPR CRISPRi Cas9 gRNA plasmids from the Qi lab CRISPR...

...Enzyme(s) Delivery Resistance Co-expressed Cas9 Depositing lab Cas9 species = S. pyogenes (PAM = NGG) 51306...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...backbone T7 BsaI In vitro transcription none, need Cas9 plasmid Chen Do you have suggestions for other plasmids...Editing Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled Libraries gRNAs Empty gRNA Vectors...

...) Validated In Resistance Co-expressed Cas9 Depositing lab Cas9 species = S. pyogenes (PAM = NGG) 42875... Publication Purify A catalytically inactive Cas9 (dCas9) can be used to purify a region of genomic DNA...immunoprecipitate FLAG-tagged Cas9. Design your gRNA sequence to direct dCas9 to a specific locus, avoiding... pneumoniae Kanamycin none, need Cas9 plasmid Marraffini 42876 pCas9 BsaI E. coli, S. pneumoniae Chloramphenicol...Plasmid Gene/Insert Promoter PI Publication Prime Edit Cas9 H840A nickase fused to a reverse transcriptase (...crRNA. This activity provides a stark contrast to Cas9 and Cpf1, which require that each DNA target have... BBa_J23119 SpeI + HindIII Ampicillin none, need Cas9 plasmid Qi Do you have suggestions for other plasmids...

...contains Cas9. ID Plasmid Expression System Promoter Guide RNA Type Cloning Enzyme Co-expressed Cas9 Selection...transcriptase (RT) is fused to the C terminus of Cas9 H840A nickase. The fusion enzyme is capable of installing...template. During prime editing, the pegRNA directs the Cas9 nickase to the target sequence where it nicks the...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...PuroTK-pegRNA_GG Mammalian, piggyBac hU6 pegRNA BsmBI Yes (Cas9 H840A + MMLV RT) PuroTK Jacob Giehm Mikkelsen 174038...Editing Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled Libraries gRNAs Empty gRNA Vectors...

...genomic deletions in mammalian cell lines using CRISPR/Cas9...and Daniel Bauer's labs details the use of CRISPR/Cas9 to create genomic deletions in mammalian cell lines...Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9. Bauer DE, Canver MC, Orkin SH. J. Vis. Exp. (95...site-specific eukaryotic genome engineering. CRISPR/Cas9 is an inexpensive, facile, and efficient genome ...required DNA yield. Perform maxi-prep for each CRISPR/Cas9 construct. Transfecting CRISPRs into Cells of Interest...NOTE: This protocol involves the delivery of CRISPR/Cas9 plasmids by electroporation 4 . This protocol is...cells. However, transient transfection of CRISPR/Cas9 plasmids may be successfully adapted to numerous...

...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... (AsCpf1 and LbCpf1) for use in mammalian cells SpCas9 variants with no detectable off-target effects ...