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CRISPR header icon CRISPR/Cas: dCas9-FokI Double-strand Break (Cut)


A catalytically inactive Cas9 (dCas9) fused to half of the FokI nuclease. When FokI dimerizes it can generate double strand breaks (DSBs, Cut) at specific sequences. Two unique gRNAs are required to target the dCas9-FokI to a region of the genome. In order for FokI to dimerize, the two halves, each guided by a unique gRNA, must bind ~30bp apart. This technique combines the reduced off-target effects of the dual-nickase system with the additional care of the dimerization of FokI.




Mammalian

ID Plasmid Gene/Insert Promoter PI Publication Hidden Extra Search Info
52970FokI-dCas9Fok1 fused to dCas9 (Other)CMV Liu Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat Biotechnol. 2014 Apr 25. doi: 10.1038/nbt.2909. Expresses Fok1 nuclease domain fused to catalytically inactive Cas9 DNA-binding domain in mammalian cells JDS246
53369pSQT1601hCsy4-T2A-NLS-hFokI-dCas9-NLSCAG Joung Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat Biotechnol. 2014 Apr 25. doi: 10.1038/nbt.2908. human codon optimized Csy4 and FokI-dCas9 expression plasmid pCAG-CFP
60901pXFokI-dCas9CBh and U6 Conklin Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing Scientific Reports 6, Article number: 23549 (2016) Dual Expression Vector for FokI-dCas9 and gRNA pUC ori vector
  • Tag / Fusion Protein
    • 3XFLAG (C terminal on backbone)
  • 63602pX330A_FokI-1x2FokI-dCas9 (Other)CBh Yamamoto Production of knockout mice by DNA microinjection of various CRISPR/Cas9 vectors into freeze-thawed fertilized oocytes. BMC Biotechnol. 2015 May 22;15(1):33. Expresses FokI-dCas9 and gRNA pUC ori vector
    63603pX330A_FokI-1x3FokI-dCas9 (Other)CBh Yamamoto Production of knockout mice by DNA microinjection of various CRISPR/Cas9 vectors into freeze-thawed fertilized oocytes. BMC Biotechnol. 2015 May 22;15(1):33. Expresses FokI-dCas9 and gRNA pUC ori vector
    63604pX330A_FokI-1x4FokI-dCas9 (Other)CBh Yamamoto Production of knockout mice by DNA microinjection of various CRISPR/Cas9 vectors into freeze-thawed fertilized oocytes. BMC Biotechnol. 2015 May 22;15(1):33. Expresses FokI-dCas9 and gRNA pUC ori vector
    63605pX330A_FokI-1x5FokI-dCas9 (Other)CBh Yamamoto Production of knockout mice by DNA microinjection of various CRISPR/Cas9 vectors into freeze-thawed fertilized oocytes. BMC Biotechnol. 2015 May 22;15(1):33. Expresses FokI-dCas9 and gRNA pUC ori vector
    63606pX330A_FokI-1x6FokI-dCas9 (Other)CBh Yamamoto Production of knockout mice by DNA microinjection of various CRISPR/Cas9 vectors into freeze-thawed fertilized oocytes. BMC Biotechnol. 2015 May 22;15(1):33. Expresses FokI-dCas9 and gRNA pUC ori vector
    63607pX330A_FokI-1x7FokI-dCas9 (Other)CBh Yamamoto Production of knockout mice by DNA microinjection of various CRISPR/Cas9 vectors into freeze-thawed fertilized oocytes. BMC Biotechnol. 2015 May 22;15(1):33. Expresses FokI-dCas9 and gRNA pUC ori vector
    85754pSH-Csy4-T2A-SpRFNCsy4-T2A-FokI-dCas9 (Other)CAG Stanton Re-engineered RNA-Guided FokI-Nucleases for Improved Genome Editing in Human Cells. Mol Ther. 2017 Feb 1;25(2):342-355. doi: 10.1016/j.ymthe.2016.11.007. Expresses S. pyogenes FokI-dCas9-NLS with a 25 amino acid linker (GGGGS)5 fusion. Also expresses Csy4 for cleavage of multiplexed gRNA transcripts. Derived from pSQT1601 (Addgene #53369). pSQT-1601
    85755pSH-Csy4-T2A-SaRFNCsy4-T2A-FokI-SadCas9 (Other)CAG Stanton Re-engineered RNA-Guided FokI-Nucleases for Improved Genome Editing in Human Cells. Mol Ther. 2017 Feb 1;25(2):342-355. doi: 10.1016/j.ymthe.2016.11.007. Expresses S.aureus FokI-dCas9-NLS with a 36 amino acid GSAT linker fusion. Also expresses Csy4 for cleavage of multiplexed gRNA transcripts. Backbone derived from pSQT1601 (Addgene #53369). pSQT-1601
    85756pSH-Csy4-T2A-SpRFN-P2A-GFP-multi-gRNACsy4-T2A-SpRFN-P2A-GFP (Other), gRNA (Synthetic)CMV, U6 Stanton Re-engineered RNA-Guided FokI-Nucleases for Improved Genome Editing in Human Cells. Mol Ther. 2017 Feb 1;25(2):342-355. doi: 10.1016/j.ymthe.2016.11.007. All in one plasmid that expresses Csy4, S.pyogenes FokI-dCas9-NLS, and GFP. Also encodes for multiplexed gRNAs. Backbone derived from pSQT1601 (Addgene #53369). pSQT-1601

    Drosophila

    ID Plasmid Gene/Insert Promoter PI Publication Hidden Extra Search Info
    62210pnos-Fok1:dCas9-nosFok1-dCas9nanos Bullock CRISPRflydesign (unpublished) Expresses Fok1:dCas9 under control of nanos promoter and 3'UTR. For germ line restricted high specificity genome engineering in Drosophila melanogaster. pnos
    62211pAct-Fok1:dCas9Fok1-dCas9act5C Bullock CRISPRflydesign (unpublished) Expresses Fok1:dCas9 under control of act5C promoter and SV40 3'UTR. For ubiquitous high specificity genome engineering in Drosophila melanogaster. pact

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