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CRISPR header icon CRISPR/Cas Plasmids: Single-strand Break (Nick)


A mutated "nickase" version of the Cas9 enzyme generates a single-strand DNA break (Nick) at a specific location based on a co-expressed gRNA-defined target sequence, rather than a double-strand DNA break (Cut) produced by the wild type enzyme. Nicks are preferentially repaired in the cell by homology directed repair (HDR), using the intact strand as the template. HDR has high fidelity and rarely results in errors. Two adjacent, opposite strand nicks can cause a double strand break (DSB) and trigger error-prone non-homologous end joining (NHEJ) repair; however, in the presence of a repair template, the double nicks can be repaired by HDR. Double nicking greatly reduces unwanted off-target effects.


Mammalian

Plasmid Gene/Insert Promoter Selectable Marker PI Publication Hidden Extra Search Info
hCas9_D10ACas9_D10ACMV Church RNA-Guided Human Genome Engineering via Cas9. Science. 2013 Jan 3. Expresses human codon optimized Cas9 D10A mutant which functions as a nickase for genome engineering pcDNA3.3-TOPO
pX334-U6-DR-BB-DR-Cbh-NLS-hSpCas9n(D10A)-NLS-H1-shorttracr-PGK-purohumanized S. pyogenes Cas9 (D10A) nickasePuromycin Zhang Multiplex Genome Engineering Using CRISPR/Cas Systems. Science. 2013 Jan 3. This plasmid separately encodes a human codon-optimized SpCas9 nickase, a tracrRNA and customizable crRNA. pUC ori vector
  • Tag / Fusion Protein
    • HA (N terminal on insert)
  • pX335-U6-Chimeric_BB-CBh-hSpCas9n(D10A)humanized S. pyogenes Cas9 (D10A) nickase Zhang Multiplex Genome Engineering Using CRISPR/Cas Systems. Science. 2013 Jan 3. A human codon-optimized SpCas9 nickase and chimeric guide RNA expression plasmid. pUC ori vector
  • Tag / Fusion Protein
    • HA (N terminal on insert)
  • pCas9D10A_GFPCas9D10A-2A-GFP (Synthetic)CAG Musunuru Cas9 GFP plamsids (unpublished) Co-expression of human codon-optimized Cas9 (D10A) mutant nickase and GFP, plasmid optimized for expression in human pluripotent stem cells pCAG
  • Tag / Fusion Protein
    • 2A-GFP (C terminal on insert)
  • pSpCas9n(BB)-2A-GFP (PX461)hSpCas9n (Synthetic)Cbh Zhang Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013 Nov;8(11):2281-308. doi: 10.1038/nprot.2013.143. Epub 2013 Oct 24. Cas9n (D10A nickase mutant) from S. pyogenes with 2A-EGFP, and cloning backbone for sgRNA PX461
    pSpCas9n(BB)-2A-Puro (PX462)hSpCas9n-2A-Puro (Synthetic)CbhPuromycin Zhang Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013 Nov;8(11):2281-308. doi: 10.1038/nprot.2013.143. Epub 2013 Oct 24. NOTE: A new version of this plasmid is now available. See Addgene plasmid 62987 PX462
    pSpCas9n(BB) (PX460)hSpCas9n (Synthetic)Cbh Zhang Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013 Nov;8(11):2281-308. doi: 10.1038/nprot.2013.143. Epub 2013 Oct 24. Cas9n (D10A nickase mutant) from S. pyogenes PX460
    pST1374-N-NLS-flag-linker-Cas9-H840ACas9 nickaseCMVBlasticidin Huang Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nat Methods. 2014 Mar 2. doi: 10.1038/nmeth.2857. Cas9 nickase optimized for nuclear import. Addgene 44758
    pST1374-N-NLS-flag-linker-Cas9-D10ACas9 nickaseCMVBlasticidin Huang Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nat Methods. 2014 Mar 2. doi: 10.1038/nmeth.2857. Cas9 nickase optimized for nuclear import. Addgene 44758
    pCAG-T3-hCasD10A-pACodon-optimized Cas9 D10A (Synthetic)CAG promoter Fujii Efficient generation of genome-modified mice via offset-nicking by CRISPR/Cas system. Biochem Biophys Res Commun. 2014 Jan 31. pii: S0006-291X(14)00176-4. doi: 10.1016/j.bbrc.2014.01.141. Expresses Cas9-nickase in mammalian cells and zygotes. pCAGGS
    pCAG-hCas9D10AhCas9D10A (Other)CAGNeomycin (select with G418) Hatada pCAG-hCas9D10A (unpublished) The expression vector for humanized nickase Cas9 (Cas9D10A) under CAG promoter pEGFP-N1
    pNW3Csy4-T2A-Cas9n (D10A)CAG Joung Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat Biotechnol. 2014 Apr 25. doi: 10.1038/nbt.2908. Csy4 and Cas9 D10A nickase expression plasmid pCAG-CFP
    N-Terminal Split Cas9 D10A Nickase with GyrA inteinD10A Nickase humanized S. pyogenes Cas9 with Gyra Nsplit Intein (Homo sapiens)CBh Bao Trans-spliced Cas9 allows cleavage of HBB and CCR5 genes in human cells using compact expression cassettes. Sci Rep. 2015 Jul 1;5:10777. doi: 10.1038/srep10777. Expresses N-terminus of D10A SpCas9 nickase domain fused to a GyrA intein, flanked by ITRs for AAV packaging. Combine with C-Terminal Split Cas9 Gyra Intein for full length SpCas9 nickase production Plasmid 42235: pX335-U6-Chimeric_BB-CBh-hSpCas9n(D10A)
    pHL-EF1a-SphcCas9(D10A)-iP-ACRISPR Cas9 D10A (Other), puromycin resistance gene (Other)Puromycin Hotta Precise Correction of the Dystrophin Gene in Duchenne Muscular Dystrophy Patient Induced Pluripotent Stem Cells by TALEN and CRISPR-Cas9. Stem Cell Reports. 2014 Nov 25. pii: S2213-6711(14)00335-X. doi: 10.1016/j.stemcr.2014.10.013. Expresses D10A mutant (nickase) of human codon-optimized Cas9 (derived from Streptococcus pyogenes) and pruomycin resistance gene. pHL-EF1a-GW-iP-A
    pXCas9H840ACBh 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 Cas9 H840A nickase and gRNA pUC ori vector
  • Tag / Fusion Protein
    • 3XFLAG (N terminal on backbone)
  • c3GIC9nhumanized S. Pyogenes Cas9 (D10A) (Other)TRE3G Dow Inducible in vivo genome editing with CRISPR-Cas9. Nat Biotechnol. 2015 Apr;33(4):390-4. doi: 10.1038/nbt.3155. Epub 2015 Feb 18. col1a1 targeting vector for inducible [TRE3G]-GFP-IRES-Cas9n (D10A variant) expression. Contains NsiI cloning site for U6-sgRNA cassettes col1a1 Flp-in targeting construct
    pSpCas9n(BB)-2A-Puro (PX462) V2.0hSpCas9n-2A-Puro (Synthetic)Puromycin Zhang Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013 Nov;8(11):2281-308. doi: 10.1038/nprot.2013.143. Epub 2013 Oct 24. Cas9n (D10A nickase mutant) from S. pyogenes with 2A-Puro, and cloning backbone for sgRNA (V2.0) PX462
    lentiCas9n(D10A)-BlastCas9 (Synthetic), Blasticidin resistanceEFS-NS, EFS-NSBlasticidin Zhang Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods. 2014 Aug;11(8):783-4. doi: 10.1038/nmeth.3047. Expresses human codon-optimized S. pyogenes Cas9n (D10A nickase) protein and blasticidin resistance from EFS promoter. Lentiviral backbone. pFUGW
    PX429 SpCas9 N863A nickaseSpCas9 N863A nicking mutant (Synthetic)Cbh Zhang Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell. 2014 Feb 27;156(5):935-49. doi: 10.1016/j.cell.2014.02.001. Epub 2014 Feb 13. SpCas9 N863A nickase PX165
    pCR1054T7pr_6xHis-MBP-TEV-SPynCas9 (D10A)-2xNLS Corn Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nat Biotechnol. 2016 Jan 20. doi: 10.1038/nbt.3481. Express Streptococcus pyogenes nCas9 (D10A) nickase carrying two C-terminal SV40 NLS SPynCas9
    pCR1055T7pr_6xHis-MBP-TEV-SPynCas9 (H840A)-2xNLS Corn Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nat Biotechnol. 2016 Jan 20. doi: 10.1038/nbt.3481. Express Streptococcus pyogenes nCas9 (H840A) nickase carrying two C-terminal SV40 NLS SPynCas9
    AIO-GFP Jackson CRISPR-Cas9(D10A) nickase-based genotypic and phenotypic screening to enhance genome editing. Sci Rep. 2016 Apr 15;6:24356. doi: 10.1038/srep24356. All-in-One plasmid encoding dual U6 promoter-driven sgRNAs and EGFP-coupled Cas9-D10A nickase to enhance efficient and accurate genome editing All-in-One
    AIO-mCherry Jackson CRISPR-Cas9(D10A) nickase-based genotypic and phenotypic screening to enhance genome editing. Sci Rep. 2016 Apr 15;6:24356. doi: 10.1038/srep24356. All-in-One plasmid encoding dual U6 promoter-driven sgRNAs and mCherry-coupled Cas9-D10A nickase to enhance efficient and accurate genome editing All-in-One
    AIO-PuroCbhPuromycin Jackson CRISPR-Cas9(D10A) nickase-based genotypic and phenotypic screening to enhance genome editing. Sci Rep. 2016 Apr 15;6:24356. doi: 10.1038/srep24356. All-in-One plasmid encoding dual U6 promoter-driven sgRNAs and Cas9-D10A nickase linked via 2A peptide with puromycin resistant marker to enhance efficient and accurate genome editing All-in-One_Nickase-D10A
  • Tag / Fusion Protein
    • Puromycin resistant marker


  • Drosophila

    Plasmid Gene/Insert Promoter Selectable Marker PI Publication Hidden Extra Search Info
    pDCC5Cas9-D10A (Synthetic)hsp70Bb Duchek Efficient CRISPR/Cas9 Plasmids for Rapid and Versatile Genome Editing in Drosophila. G3 (Bethesda). 2014 Sep 17. pii: g3.114.014126. doi: 10.1534/g3.114.014126. Bi-cistronic Drosophila CRISPR/Cas9 vector, contains hsp70>Cas9-D10A nickase and U6>sgRNA cassette pHW


    Plant

    Plasmid Gene/Insert Promoter Selectable Marker PI Publication Hidden Extra Search Info
    pBUN501zCas9D10A (Synthetic), gRNA scaffold (Synthetic)Ubi1p, AtU6-26pBar Chen A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol. 2014 Nov 29;14(1):327. CRISPR/Cas based plant genome editing and gene regulation; expresses zCas9D10A, gRNA scaffold for insertion of target sequence (AtU6-26 promoter), Bar resistance pGreen-like binary vector
    pHSN501zCas9D10A (Synthetic), gRNA scaffold (Synthetic)2×35Sp, AtU6-26pHygromycin Chen A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol. 2014 Nov 29;14(1):327. CRISPR/Cas based plant genome editing and gene regulation; expresses zCas9D10A, gRNA scaffold for insertion of target sequence (AtU6-26 promoter), Hyg resistance pGreen-like binary vector
    pYPQ159hSpCas9D10A (Human codon optimized) (Other) Qi A CRISPR/Cas9 toolbox for multiplexed plant genome editing and transcriptional regulation. Plant Physiol. 2015 Aug 21. pii: pp.00636.2015. Gateway entry vector with hSpCas9D10A pNJB185-linker6
    pDGE76pnos:nptII-tmos (Other), p2x35S:Cas9 D10A-tnos (Other), BsaI-ccdB_CmR-BsaI Stuttmann Generation of chromosomal deletions in dicotyledonous plants employing a user-friendly genome editing toolkit. Plant J. 2016 Aug 31. doi: 10.1111/tpj.13319. recipient plasmid [multiplexing, nickase]; p35S:Cas9 D10A, nptII pVM_BGW
    pDGE77pnos:PAT-tnos (Other), p2x35S:Cas9 D10A-tnos (Other), BsaI-ccdB_CmR-BsaI Stuttmann Generation of chromosomal deletions in dicotyledonous plants employing a user-friendly genome editing toolkit. Plant J. 2016 Aug 31. doi: 10.1111/tpj.13319. recipient plasmid [multiplexing, nickase]; p35S:Cas9 D10A, BASTA pVM_BGW
    pDGE78pnos:nptII-tmos (Other), pPcUbi:Cas9 D10A-tnos (Other), BsaI-ccdB_CmR-BsaI Stuttmann Generation of chromosomal deletions in dicotyledonous plants employing a user-friendly genome editing toolkit. Plant J. 2016 Aug 31. doi: 10.1111/tpj.13319. recipient plasmid [multiplexing, nickase]; pPcUbi:Cas9 D10A, nptII pVM_BGW
    pDGE79pnos:PAT-tnos (Other), pPcUbi:Cas9 D10A-tnos (Other), BsaI-ccdB_CmR-BsaI Stuttmann Generation of chromosomal deletions in dicotyledonous plants employing a user-friendly genome editing toolkit. Plant J. 2016 Aug 31. doi: 10.1111/tpj.13319. recipient plasmid [multiplexing, nickase]; pPcUbi:Cas9 D10A, BASTA pVM_BGW
    pTX179Hygromycin Voytas A Single Transcript CRISPR-Cas9 System for Efficient Genome Editing in Plants. Mol Plant. 2016 Jul 6;9(7):1088-91. doi: 10.1016/j.molp.2016.05.001. Epub 2016 May 19. Express STU (Single Transcriptional Unit) Cas9 nickase with Maize Ubiquitin1 promoter in plants pTX172
  • Tag / Fusion Protein
    • SV40 NLS (C terminal on insert)


  • Bacteria

    Plasmid Gene/Insert Promoter Selectable Marker PI Publication Hidden Extra Search Info
    pMJ825Cas9T7 Doudna A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science. 2012 Jun 28. pEC-K-MBP
  • Tag / Fusion Protein
    • His6 (N terminal on backbone)
  • pMJ826Cas9T7 Doudna A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science. 2012 Jun 28. pEC-K-MBP
  • Tag / Fusion Protein
    • His6 (N terminal on backbone)
  • pMCSG7-D16A-NmeCas9pMCSG7-D16A-NmeCas9 (Other) Sontheimer DNase H Activity of Neisseria meningitidis Cas9. Mol Cell. 2015 Oct 15;60(2):242-55. doi: 10.1016/j.molcel.2015.09.020. bacterial pMCSG7 expression vector expressing Nme cas9 nickase with a D16A mutation (RuvC domain) with T7 promoter, N-terminal His tag and TEV site pMCSG7
    pMCSG7-H588A-NmeCas9pMCSG7-H588A-NmeCas9 (Other)T7 Sontheimer DNase H Activity of Neisseria meningitidis Cas9. Mol Cell. 2015 Oct 15;60(2):242-55. doi: 10.1016/j.molcel.2015.09.020. bacterial pMCSG7 expression vector expressing Nme cas9 nickase with H588A mutation (HNH domain) with T7 promoter, N-terminal His tag and TEV site pMCSG7
    pNICKclos2.0Cas9 nickase (Other), sgRNA to xylR (Synthetic)Pthl, Pj23119Erythromycin in clostridium Yang CRISPR-based genome editing and expression control systems in Clostridium acetobutylicum and Clostridium beijerinckii. Biotechnol J. 2016 May 23. doi: 10.1002/biot.201600053. Genome editing for gene xylR (cbei-2385) in clostridium beijerinckii NCIMB 8052 pXY1
    pNICKclos1.0Cas9 nickase (Other), sGRNA to pyrEptb, j23119Erythromycin in clostridium Yang CRISPR-based genome editing and expression control systems in Clostridium acetobutylicum and Clostridium beijerinckii. Biotechnol J. 2016 May 23. doi: 10.1002/biot.201600053. Genome editing for gene pyrE (CAC-002) in Clostridium acetobutylicum ATCC 824 pIMP1-ptb
    pCas9(D10A)Cas9 D10A (Synthetic)pTeto Wang Targeted Large-Scale Deletion of Bacterial Genomes Using CRISPR-Nickases. ACS Synth Biol. 2015 Nov 20;4(11):1217-25. doi: 10.1021/acssynbio.5b00132. Epub 2015 Oct 25. Nicking Cas9 derived from pWT Cas9 (Addgene #44250). Ampicillin resistance marker, the tetracycline repressor (TetR), a ColE1 origin of replication and Streptococcus pyogenes Cas9 D10A pWTCas9


    Yeast

    ID Plasmid Purpose Gene/Insert Promoter Selectable Marker PI Publication Hidden Extra Search Info
    79616pRS415_pGal-nCas9(H840A)Expresses nCas9(H840A) in yeast cellsSpCas9 (Other)pGal1LEU2 Kondo Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science. 2016 Aug 4. pii: aaf8729. Expresses nCas9(H840A) in yeast cells pRS415
    79617pRS315e_pGal-nCas9(D10A)-PmCDA1Expresses nCas9(D10A)-PmCDA1 in yeast cellsSpCas9 (Other)pGal1LEU2 Kondo Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science. 2016 Aug 4. pii: aaf8729. Expresses nCas9(D10A)-PmCDA1 in yeast cells pRS315
    79618pRS415_pGal-nCas9(D10A)Expresses nCas9(D10A) in yeast cellsSpCas9 (Other)pGal1LEU2 Kondo Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science. 2016 Aug 4. pii: aaf8729. Expresses nCas9(D10A) in yeast cells pRS415