...editors are available with multiple Cas9 variants including high fidelity Cas9’s. Further advancements have ...are created by fusing Cas9 nickase or catalytically inactive “dead” Cas9 (dCas9) to a cytidine deaminase...base editors, the evolved TadA domain is fused to a Cas9 protein to create the adenine base editor. Both ...fusions, modifying the linker region between Cas variant and deaminase to adjust the editing window, or ...systems create a wide spectrum of single-nucleotide variants (somatic hypermutation) in a wider editing window... 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...

... available with multiple Cas9 variants, including high fidelity Cas9s, Cas9s with various PAM requirements... NAG binding) SpCas9 VRER variant 3' NGCG SpCas9 EQR variant 3' NGAG SpCas9 VQR variant 3' NGAN or NGNG...Species/Variant of Cas9 PAM Sequence* Streptococcus pyogenes (SP); SpCas9 3' NGG SpCas9 D1135E variant 3' ... engineering. Read more about Cas9 variants . Anti-CRISPR Prolonged Cas9 activity can increase the risk...LbCpf1) 5' TTTV AsCpf1 RR variant 5' TYCV LbCpf1 RR variant 5' TYCV AsCpf1 RVR variant 5' TATV Campylobacter...strand SpCas9-HF1 - disrupt Cas9’s interactions with DNA phosphate backbone HypaCas9 - increase Cas9 proofreading...PAM-flexible or PAMless Cas9s. SpCas9-derived variants with non-NGG PAM sequences include: xCas9 - NG, GAA, and...

...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 Modified...

...fusion for gene activation CRISPR/Cas9 Plasmid Variants Figure 1: Schematic of the CRISPR Collection. Plasmids...in retinal development the lab utilizes a CRISPR/Cas9 based approach to knockout, repress, and activate...taken advantage of the Zhang lab all-in-one (guide/Cas9/reporter) pX458 plasmid family. They have made several... with EF1alpha addition of an mCherry reporter variant nuclear reporters via an H2B fusion shuttle plasmid... plasmids dCas9-KRAB-MeCP2 fusion for gene and non-coding enhancer sequence repression dCas9-VPR fusion...175574 express dCas9-KRAB-MeCP2 for repression. Plasmids 175571 and 175572 express dCas9-VPR for activation...Sources Original pX458 backbone ( Addgene #48138 ) dCas9 + KRAB-MeCP2 ( Addgene #110821 ) VPR ( Addgene #63798...

...single species or PAM binding variant of Cas9. For instance, wild-type SpCas9 must be used with targets that...increases off-target effect. Which species or variant of Cas9 ( S. pyogenes , S. aureus etc.) was this gRNA...Editing Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled Libraries gRNAs Empty gRNA Vectors...

...protein variant (CyPet) optimized for FRET measurements with a yellow fluorescent protein variant (YPet)...protein variant (YPet) optimized for FRET measurements with a cyan fluorescent protein variant (CyPet)...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... periplasm. Series contains vectors with a YFP variant that functions in the periplasm and can be co-localized... pCri System Various Plasmids T7/lacO and lacO variants that can be expressed in B. subtilis Various combinations...Anhydrotetracycline inducible expression of catalytically inactive Cas9 ( S. pyogenes ) which can be combined with expression...

...of plasmids expressing different functional variants of Cas9 and empty gRNA backbones. A few examples of... 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...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...

...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...want to consider alternative Cas enzymes ( see Cas9 variants and PAM sequences ). Once possible PAM sequences...and/or Cas9 expression or (2) a lack of efficient target cleavage in cells expressing both Cas9 and gRNA...

...a single species or PAM binding variant of Cas9 (e.g. Wild-type SpCas9 must be used with targets that ...activity. Know your target. Which species or variant of Cas9 ( S. pyogenes, S. aureus etc.) was this gRNA...C. elegans TCCGCTACCATAGGCACCA 71479 cut VQR Cas9 variant 26680661 Fire dpy‐10 C. elegans GCTACCATAGGCACCACGAG...and Resources page have been used to indicate the Cas9 application the gRNA was designed to accomplish....Target Species Target Sequence Plasmid ID Application Cas9 Species PubMed ID Depositor OCT4 H. sapiens CTCCCATGCATTCAAACTG...10 C. elegans CGCTACCATAGGCACCACG 71516 cut VRER Cas9 mutant 26680661 Fire dpy-10 C.elegans GCTACCATAGGCACCACGAG...Christiaen EGFP A. victoria multiple, see article 60071 dCas9-FokI S. pyogenes 24770325 Joung EGFP A. victoria...

...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...

... 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... into one vector, and plasmids that provide Cas9 variants. All components are then assembled into a single...

...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...sgRNA expression hSyn AAV Zhang 60820 pSECC Cre, Cas9 and sgRNA expression EF-1 alpha Lentiviral Jacks...6xHis Cre fused to supernegatively charged GFP variant T7 Bacterial Liu 62657 pAYC iCre CAG Mammalian ...9xGGS-Cre-6xHis Cre fused to highly cationic GFP variant T7 Bacterial Liu 62939 pET-Cre-6xHis Cre-6xHis ...