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CRISPR: dCas9-FokI


A catalytically inactive Cas9 (dCas9) is fused to FokI nuclease. When FokI dimerizes, it generates a double-strand break (DSB) at a specific sequence. Two unique gRNAs, binding ~15-25 bp apart, are required for dCas9-FokI to dimerize at a given region of the genome. Like nickase technology, this technique reduces unwanted off-target effects of CRISPR.

CRISPR-FokI-horizontal.png

Browse, sort, or search the tables below for CRISPR-FokI plasmids.
Plasmids are available for expression in mammalian systems and Drosophila.


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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