...Technique PI Purpose pCas9 42876 CRISPR Luciano Marraffini Bacterial expression of Cas9 nuclease gRNA. For...Anhydrotetracycline inducible expression of wild-type Cas9 from S. pyogenes for inducing double stranded breaks. For...efficient enough that selection is not required. pET-HIS-Sangamo 40786 TALEN Gang Bao Gateway cloning .... coli codon optimized FokI endonuclease domain. pET-Sangamo-His 40787 TALEN Gang Bao Gateway cloning ...terminal, TEV cleavable 6x-His tag Ophear Gileadi pET-28-a based expression vector containing N-terminal...various epitope tags useful for protein purification. pET LIC Series Various Plasmids T7-lacO (lactose/IPTG...Anhydrotetracycline inducible expression of catalytically inactive Cas9 ( S. pyogenes ) which can be combined with expression...

...Cre-dependent Cas9: Cre-dependent Cas9 can be knocked in to a mouse at the Rosa26 locus to facilitate Cas9 mediated...iRFP670, Cre, and GFP EFS Lentiviral Jacks 68468 Cas9-2A-Cre Cas9 and Cre CAG Mammalian Zhang 68477 pTRE:iRFP670...CMV Mammalian Sato 123128 pET-His-Cre 6xHis-Cre T7 Bacterial Liu 123131 pET-His-VCre 6xHis-VCre T7 Bacterial...sgRNA expression hSyn AAV Zhang 60820 pSECC Cre, Cas9 and sgRNA expression EF-1 alpha Lentiviral Jacks...Targeting vector Mouse Nxph4 Mammalian Zeng 62372 pET-(-30)GFP-9xGGS-Cre-6xHis Cre fused to supernegatively...62730 CRE recombinase Cre T7 Bacterial Geijsen 62938 pET-(pos36)GFP-9xGGS-Cre-6xHis Cre fused to highly cationic...cationic GFP variant T7 Bacterial Liu 62939 pET-Cre-6xHis Cre-6xHis T7 Bacterial Liu 63704 pRetroX GFP...

... 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...

...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...

...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...Gonzales AP, Li Z, Peterson RT, Yeh JR, Aryee MJ, Joung JK. 2015. Engineered CRISPR-Cas9 nucleases with altered...the crRNA would bind. (5) After the complex binds, Cas9 separates the double stranded DNA target and cleaves...

...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...

... to immunoprecipitate FLAG-tagged Cas9. In the CAPTURE system, Cas9 is tagged with a biotin acceptor site... Publication Purify A catalytically inactive Cas9 (dCas9) can be used to purify a region of genomic DNA...Publication Visualize A catalytically inactive Cas9 (dCas9) fused to a fluorescent protein (FP) can help...Promoter Selectable Marker Publication Prime Edit Cas9 H840A nickase fused to a reverse transcriptase (...PI Publication Interfere Catalytically dead dCas9, or dCas9 fused to a transcriptional repressor peptide...Editing Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled Libraries gRNAs Empty gRNA Vectors... CRISPR Blog Posts Base Edit Catalytically dead dCas9 fused to a cytidine deaminase protein becomes a ...

... inactive Cas9 (dCas9) fused to a fluorescent marker like GFP, researchers have turned dCas9 into a customizable...has been used for dynamic tracking of repetitive and non-repetitive genomic loci, as well as chromosome ...Editing Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled Libraries gRNAs Empty gRNA Vectors...given region. For example, chromosome-specific repetitive loci can be efficiently visualized in living ... targeted sequences in close proximity. A non-repetitive genomic locus can also be labeled by co-delivering...

...Gideon Dreyfuss 71875 pET-Sac-Abeta(M1-42) APP T7 Alzheimer's Dominic Walsh 71876 pET-Sac-Abeta(M1-40) APP...Bruce Beutler 127151 pET-Sac-Abeta(MC1-42) APP T7 Alzheimer's James Nowick 127152 pET-Sac-Abeta(MC1-40) ... 190191 pET-Duet1-His-TEV-mCherry-OPTN WT OPTN His, TEV, mCherry ALS Sascha Martens 190192 pET-Duet1-6xHis-TEV-OPTN...sgHTT1-7sk-sgCas9 HTT U6, 7sk Huntington's Nicole Deglon 190901 pAAVss-U6-sgHTT51-7sk-Cas9 HTT U6, 7sk...GFP CMV Spinal muscular atrophy Greg Matera 38005 pET-21a(+)/MBP-His-PINK1 PINK1 His, MBP T7 Parkinson'...IDR-mCherry-Cry2-NLS TBP Parkinson's Denes Hnisz 145283 pET-45b-TBP(38Q)-IDR-mCherry TBP Parkinson's Denes Hnisz...CMV Ataxia telangiectasia Charles Swanton 173759 pET-Sac-Abeta(M1-42/C18C33) APP T7 Alzheimer's James ...

...resources for a full selection of plasmids expressing Cas9 and empty gRNA backbones. A few examples of additional...spoken language and hand skills, development of repetitive hand stereotypies, and gait dysfunction or absence...