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CRISPR header icon CRISPR/Cas9 Plasmids for use in Plants

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Addgene is working with the leading scientists in the field to assemble the reagents and information you need to use the CRISPR/Cas9 technology in your own lab. The following Cas9 and gRNA expression plasmids have been designed for use in Plants.

Cut

Fully functional Cas9 enzymes designed to introduce a double-strand break (DSBs) at a specific location based on a co-expressed gRNA-defined target sequence. DSBs are preferentially repaired in the cell by non-homologous end joining (NHEJ); a mechanism which frequently causes insertions or deletions (InDels) in the DNA, possibly resulting in frameshifts. If a repair template with high homology to the DNA surrounding the DSB is introduced along with the Cas9 and gRNA plasmids, the cell may instead repair the break using homology-directed repair (HDR). HDR is much less error-prone than NHEJ and can be used to faithfully introduce specific genomic changes.

Plasmid Gene/Insert Promoter Selectable Marker PI Publication Hidden Extra Search Info
pK7WGF2::hCas9hCas9 (Synthetic)35SKanamycin Kamoun Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nat Biotechnol. 2013 Aug 8;31(8):691-3. doi: 10.1038/nbt.2655. Expesses the human codon usage Cas9 nuclease (Mali et al. Science 339, 823-826, 2013) with an N-terminal GFP tag from the 35S promoter in the plant tissue pK7WGF2
pICH41308::hCas9hCas9 (Synthetic) Kamoun Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system. Plant Methods. 2013 Oct 11;9(1):39. Level 0 hCas9 module pICH41308
pICH47742::2x35S-5’UTR-hCas9(STOP)-NOST35Sp::hCas9 (Synthetic) Kamoun Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system. Plant Methods. 2013 Oct 11;9(1):39. Level 1 hCas9 module pICH47742
pAGM4723::AtU6p::sgRNA2-2x35S-5′UTR::Cas9::NOST-AtU6p::sgRNA1sgRNA_PDS2-Cas9-sgRNA_PDS1 (Synthetic) Kamoun Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system. Plant Methods. 2013 Oct 11;9(1):39. Level 2 construct pAGM4723
pBUN4113×FLAG-NLS-zCas9-NLS (Synthetic), gRNA scaffold (Synthetic)Ubi1p, OsU3pBar 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 3×FLAG-NLS-zCas9-NLS, gRNA scaffold for insertion of target sequence (OsU3 promoter), Bar resistance pGreen-like binary vector
pHSN4013×FLAG-NLS-zCas9-NLS (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 3×FLAG-NLS-zCas9-NLS, gRNA scaffold for insertion of target sequence (AtU6-26 promoter), Hyg resistance pGreen-like binary vector
pRGE31Rice snoRNA U3 and dual 35S promoter Yang RNA-Guided Genome Editing in Plants Using a CRISPR-Cas System. Mol Plant. 2013 Nov;6(6):1975-83. doi: 10.1093/mp/sst119. Epub 2013 Aug 17. Expressing sgRNA and Cas9 in plants. The sgRNA was controlled by rice snoRNA U3 promoter pUGW11
pRGEB31Rice snoRNA U3 and dual 35S promoterHygromycin Yang RNA-Guided Genome Editing in Plants Using a CRISPR-Cas System. Mol Plant. 2013 Nov;6(6):1975-83. doi: 10.1093/mp/sst119. Epub 2013 Aug 17. Binary vector derived from pRGE31. Deliver sgRNA and Cas9 into plant. by agrobacterium mediated transformation. pCAMBIA1300
HBT-pcoCas9pcoCas9 (Synthetic)hybrid constitutive promoter 35SPPDK Sheen Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat Biotechnol. 2013 Aug;31(8):688-91. doi: 10.1038/nbt.2654. transient expression of pcoCas9 gene in plant cells HBT-FLAG
pFGC-pcoCas935SPPDK:pcoCas9:NOS cassette (Synthetic)constitutive 35SPPDK promoterBasta Sheen Sheen lab CRISPR plasmids (unpublished) A binary plasmid containing 35SPPDK:pcoCas9:NOS cassette for plant expression of Cas9 and multiple cloning sites for insertion of guide RNA pFGC-RCS
pJIT163-2NLSCas9Cas9 (Other)2x35S Gao Targeted genome modification of crop plants using a CRISPR-Cas system. Nat Biotechnol. 2013 Aug;31(8):686-8. doi: 10.1038/nbt.2650. Expression of rice codon-optimized Cas9 in plant cells pJIT163
p201N Cas9Cas9 (Synthetic), nptII (Other)2x35S, StUbi-3PG418, kanamycin Parrott Targeted genome modifications in soybean with CRISPR/Cas9. BMC Biotechnology. 2015;15:16 Cas9 driven by double 35S, nptII for plant selection, I-PpoI site to accept gRNA from pUC gRNA Shuttle pPZP
p201H Cas9Cas9 (Synthetic), hph (Other)2x35S, StUbi-3PHygromycin Parrott Targeted genome modifications in soybean with CRISPR/Cas9. BMC Biotechnology. 2015;15:16 Cas9 driven by double 35S, hygromycin resistance for plant selection, I-PpoI site to accept gRNA from pUC gRNA Shuttle pPZP
p201B Cas9Cas9 (Synthetic), BAR (Other)2x35S, 2x35SBasta Parrott Targeted genome modifications in soybean with CRISPR/Cas9. BMC Biotechnology. 2015;15:16 Cas9 driven by double 35S, BAR for plant selection, I-PpoI site to accept gRNA from pUC gRNA Shuttle pPZP
p201G Cas9Cas9 (Synthetic), sGFP (Synthetic)2x35S, 2x35SGFP Parrott Targeted genome modifications in soybean with CRISPR/Cas9. BMC Biotechnology. 2015;15:16 Cas9 driven by double 35S, GFP for plant selection, I-PpoI site to accept gRNA from pUC gRNA Shuttle pPZP
Cas9 MDC123Glycine Max codon optimized Cas9 (Other)2X35SBasta Stupar CRISPR/Cas mutagenesis of soybean and Medicago truncatula using a new web-tool and a modified Cas9 enzyme. GM Crops Food. 2015;6(4):243-52. doi: 10.1080/21645698.2015.1106063. 2x35S promoting Glycing Max codon optimized Cas9 with bar plant selection pMDC123
Cas9 MDC32Glycine Max codon optimized Cas9 (Other)2X35SHygromycin Stupar CRISPR/Cas mutagenesis of soybean and Medicago truncatula using a new web-tool and a modified Cas9 enzyme. GM Crops Food. 2015;6(4):243-52. doi: 10.1080/21645698.2015.1106063. 2x35S promoting Glycing Max codon optimized Cas9 with hygromycin plant selection pMDC32
G10 Cas9 MDC123Glycine Max codon optimized Cas9 (Other)G10Basta Stupar CRISPR/Cas mutagenesis of soybean and Medicago truncatula using a new web-tool and a modified Cas9 enzyme. GM Crops Food. 2015;6(4):243-52. doi: 10.1080/21645698.2015.1106063. G10 promoting Glycing Max codon optimized Cas9 (N and C terminus NLS) with bar plant selection pMDC123
pBUE411gRNA scaffold (Synthetic), zCas9 (Other)OsU3, UbiBasta 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 3×FLAG-NLS-zCas9-NLS, gRNA scaffold for insertion of target sequence (OsU3 promoter), Bar resistance pCambia
pHSE401gRNA scaffold (Synthetic), zCas9 (Other)AtU6-26p, 35SHygromycin 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 3×FLAG-NLS-zCas9-NLS, gRNA scaffold for insertion of target sequence (AtU6-26 promoter), Hyg resistance pCambia
pKSE401gRNA scaffold (Synthetic), zCas9 (Other)AtU6-26p, 35SNeomycin (select with G418) 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 3×FLAG-NLS-zCas9-NLS, gRNA scaffold for insertion of target sequence (AtU6-26 promoter), Kan resistance pCambia
pHUE411gRNA scaffold (Synthetic), zCas9 (Other)OsU3, UbiHygromycin 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 3×FLAG-NLS-zCas9-NLS, gRNA scaffold for insertion of target sequence (OsU3 promoter), Hyg resistance pCambia
pBUN421gRNA scaffold (Synthetic), zCas9 (Other)TaU3, UbiBasta 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 3×FLAG-NLS-zCas9-NLS, gRNA scaffold for insertion of target sequence (TaU3 promoter), Bar resistance pGreen-like
pRGEB32Rice snoRNA U3 promoter for gRNA/PTG expression and UBI promoter for Cas9 expressionHygromycin Yang Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc Natl Acad Sci U S A. 2015 Mar 17;112(11):3570-5. doi: 10.1073/pnas.1420294112. Epub 2015 Mar 2. Express sgRNA/PTG with rice snoRNA U3 promoter and Cas9 with rice ubiquitin promoter for Agrobacterium-mediated transformation. pRGEB31
pRGE32Rice snoRNA U3 promoter for gRNA/PTG expression and UBI promoter for Cas9 expression Yang Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc Natl Acad Sci U S A. 2015 Mar 17;112(11):3570-5. doi: 10.1073/pnas.1420294112. Epub 2015 Mar 2. Express sgRNA/PTG with rice snoRNA U3 promoter and Cas9 with rice ubiquitin promoter for transient expression in protoplast pRGE31
pYLCRISPR/Cas9Pubi-HCas9 (Other)maize ubiquitin promoter (Pubi)Hygromycin Liu A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant. 2015 Apr 24. pii: S1674-2052(15)00204-X. doi: 10.1016/j.molp.2015.04.007. expression of Cas9 in plants, maize ubiquitin promoter (Pubi), hygro selection pCAMBIA1300
pYLCRISPR/Cas9Pubi-BCas9 (Other)maize ubiquitin promoter (Pubi)Basta Liu A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant. 2015 Apr 24. pii: S1674-2052(15)00204-X. doi: 10.1016/j.molp.2015.04.007. expression of Cas9 in plants, maize ubiquitin promoter (Pubi), basta selection pCAMBIA1300
pYLCRISPR/Cas9P35S-BCas9 (Other)cauliflower mosaic virus 35S promoter (P35S)Basta Liu A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant. 2015 Apr 24. pii: S1674-2052(15)00204-X. doi: 10.1016/j.molp.2015.04.007. expression of Cas9 in plants, cauliflower mosaic virus 35S promoter (P35S), basta selection pCAMBIA1300
pEGB2alpha2 35s:hCas9:tNos (GB0639)hCas9 (Other)35S Orzaez GoldenBraid 2.0: a comprehensive DNA assembly framework for plant synthetic biology. Plant Physiol. 2013 Jul;162(3):1618-31. Transcriptional unit for (human codon optimized) Cas9 plant expression driven by the 35S promoter pDGB2alpha2
pICSL11056Promoter/5UTR: ZmUbi + CDS:SpCas9 + 3UTR/terminator:35s (Other) Patron Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease. Genome Biol. 2015 Nov 30;16(1):258. doi: 10.1186/s13059-015-0826-7. Level 1 Golden Gate Cassette: Cas9 expression cassette for monocotyledonous plants pICH47742 (AddGene #47742)
pICSL11060Promoter/5UTR: CsVMV+ CDS:SpCas9 + 3UTR/terminator:35s (Other) Patron Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease. Genome Biol. 2015 Nov 30;16(1):258. doi: 10.1186/s13059-015-0826-7. Level 1 Golden Gate Cassette: Cas9 expression cassette for plants (exemplified in Brassica oleracea) pICH47742 (AddGene #47742)
pYPQ150pcoCas9 (plant 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 pcoCas9 (Plant codon optimized) pNJB91
pYPQ154AteCas9 (Arabidopsis 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 AteCas9 (Arabidopsis codon optimized) pYPQ185-linker2
pYPQ158hSpCas9 (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 hSpCas9 (Human codon optimized) pNJB185-linker6
pYPQ167Cas9p (Plant codon-optimized; high GC content at 5 prime region) (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 Cas9p pYPQ185-linker2
pTC217Nuclease (Cas9/sgRNA) + Donor + GVR (Other)kanamycin Voytas High-frequency, precise modification of the tomato genome. Genome Biol. 2015 Nov 6;16(1):232. doi: 10.1186/s13059-015-0796-9. Cas9/sgRNA targeting ANT1 locus, donor molecule with 5' homology arm-Pnos:NptII-35S:ANT13' homology arm as a Gemini Viral Replicon based on Bean Yellow Dwarf Virus; sgRNA= 1b pLSLR
pTC223Nuclease (Cas9/sgRNA) + Donor + GVR (Other)kanamycin Voytas High-frequency, precise modification of the tomato genome. Genome Biol. 2015 Nov 6;16(1):232. doi: 10.1186/s13059-015-0796-9. Cas9/sgRNA targeting ANT1 locus, donor molecule with 5' homology arm-Pnos:NptII-35S:ANT13' homology arm as a Gemini Viral Replicon based on Bean Yellow Dwarf Virus; sgRNA= 7 pLSLR
pHEE401sgRNA scaffold (Synthetic), zCas9 (Other)U6-26p Arabidopsis U6 gene promoter, EC1.2 enhancer fused to EC1.1 promoterHygromycin Chen Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation. Genome Biol. 2015 Jul 21;16:144. doi: 10.1186/s13059-015-0715-0. Egg cell-specific promoter-controlled expression of 3×FLAG-NLS-zCas9-NLS. Contains gRNA scaffold for insertion of target sequence (U6-26 promoter), Hyg resistance pCambia
pHEE401EgRNA scaffold (Synthetic), zCas9 (Other)U6-26p Arabidopsis U6 gene promoter, EC1.2 enhancer fused to EC1.1 promoterHygromycin Chen Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation. Genome Biol. 2015 Jul 21;16:144. doi: 10.1186/s13059-015-0715-0. Egg cell-specific promoter-controlled expression of 3×FLAG-NLS-zCas9-NLS. Contains gRNA scaffold for insertion of target sequence (U6-26 promoter), Hyg resistance pCambia
pHEE2E-TRIsgRNA targeting TRY and CPC genes (Synthetic), sgRNA targeting ETC2 gene (Synthetic), zCas9 (Other)U6-26p Arabidopsis U6 gene promoter, U6-26p Arabidopsis U6 gene promoter, EC1.2 enhancer fused to EC1.1 promoterHygromycin Chen Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation. Genome Biol. 2015 Jul 21;16:144. doi: 10.1186/s13059-015-0715-0. Egg cell-specific promoter-controlled expression 3×FLAG-NLS-zCas9-NLS and two sgRNAs targeting three genes: ETC2, TRY, and CPC pCambia
pMpGE010Hygromycin Kohchi pMpGE010 (unpublished) For CRISPR/Cas9 genome editing in Marchantia polymorpha pMpGWB103
pMpGE011Chlorsulfuron Kohchi pMpGE010 (unpublished) For CRISPR/Cas9 genome editing in Marchantia polymorpha pMpGWB303
pEGB2Ω2_SF-35s:hCas9:tNos (GB1103)hCas9 (Other)35S Orzaez A modular toolbox for gRNA-Cas9 genome engineering in plants based on the GoldenBraid standard. Plant Methods. 2016 Feb 1;12:10. doi: 10.1186/s13007-016-0101-2. eCollection 2016. Transcriptional unit for (human codon optimized) Cas9 plant expression driven by the 35S promoter. Specially conceived to be linked with the TU of the kanamycin resistance gene GB1181. pDGB2omega2
pBAtCU6Basta Kim A simple, flexible and high-throughput cloning system for plant genome editing via CRISPR-Cas system. J Integr Plant Biol. 2016 Mar 4. doi: 10.1111/jipb.12474. Plant expression of Cas9 and gRNA empty backbone; Basta resistance pB2GW7
  • Tag / Fusion Protein
    • NLS-HA (C terminal on insert)
    		•
    pHAtCU6Hygromycin Kim A simple, flexible and high-throughput cloning system for plant genome editing via CRISPR-Cas system. J Integr Plant Biol. 2016 Mar 4. doi: 10.1111/jipb.12474. Plant expression of Cas9 and gRNA empty backbone; Hygro Resistance pH2GW7
    pHDE-35S-Cas9-mCherryHygromycin ; mCherry fluorescence Zhao An effective strategy for reliably isolating heritable and Cas9-free Arabidopsis mutants generated by CRISPR/Cas9-mediated genome editing. Plant Physiol. 2016 May 15. pii: pp.00663.2016. For CRISPR/Cas9 mediated gene editing in Arabidopsis; provides a visual screen for Cas9-free plants pHDE
    pHDE-35S-Cas9-mCherry-UBQHygromycin ; mCherry fluorescence Zhao An effective strategy for reliably isolating heritable and Cas9-free Arabidopsis mutants generated by CRISPR/Cas9-mediated genome editing. Plant Physiol. 2016 May 15. pii: pp.00663.2016. For CRISPR/Cas9 mediated gene editing in Arabidopsis; provides a visual screen for Cas9-free plants. Enable production of two gRNAs pHDE
    pKIR1.1human-codon-optimized SpCas9 (Other)AtRPS5AHygromycin ; TagRFP in seeds Higashiyama pKAMA-ITACHI vectors for highly efficient CRISPR/Cas9-mediated gene knockout in Arabidopsis thaliana. Plant Cell Physiol. 2016 Nov 17. pii: pcw191. CRISPR/Cas9 in Arabidopsis with seed a fluorescent reporter pFAST-R01
    pKI1.1human-codon-optimized SpCas9 (Other)AtRPS5A Higashiyama pKAMA-ITACHI vectors for highly efficient CRISPR/Cas9-mediated gene knockout in Arabidopsis thaliana. Plant Cell Physiol. 2016 Nov 17. pii: pcw191. CRISPR/Cas9 for Arabidopsis in pENTR/D-TOPO pENTR/D-TOPO
    pKI1.1Rhuman-codon-optimized SpCas9 (Other)AtRPS5AHygromycin ; TagRFP in seeds Higashiyama pKAMA-ITACHI vectors for highly efficient CRISPR/Cas9-mediated gene knockout in Arabidopsis thaliana. Plant Cell Physiol. 2016 Nov 17. pii: pcw191. CRISPR/Cas9 in Arabidopsis with seed a fluorescent reporter. pFAST-R01
    pAGM4723:TpCC_UreaseCas9 (Other), Urease-targeting gRNA 1 (Other), Urease-targeting gRNA 2 (Other)FCP, U6, U6Nourseothricin (Nat) Mock Editing of the urease gene by CRISPR-Cas in the diatom Thalassiosira pseudonana Plant Methods 2016, 12:49 Golden Gate Level 2 construct encoding Cas9-YFP and 2 urease-targeting gRNAs pAGM4723
    pICH47742:FCP:Cas9YFPCas9 (Other)FCP Mock Editing of the urease gene by CRISPR-Cas in the diatom Thalassiosira pseudonana Plant Methods 2016, 12:49 Golden Gate Level 1 cassette encoding a Cas9-YFP fusion under the FCP promoter pICH47742
    pKIR1.0human-codon-optimized SpCas9 (Other)AtRPS5ATagRFP in seeds Higashiyama pKAMA-ITACHI vectors for highly efficient CRISPR/Cas9-mediated gene knockout in Arabidopsis thaliana. Plant Cell Physiol. 2016 Nov 17. pii: pcw191. CRISPR/Cas9 in Arabidopsis with seed a fluorescent reporter. pFAST-R01
    pUB-Cas9Cas9 (Synthetic)UBIQUITIN10Hygromycin Weber Weber Lab CRISPR plasmids (unpublished) For ubiquitous expression of Cas9 in planta. sgRNA can be introduced as described in Hahn et al. (2017) pKB65
    [email protected]sgRNA against GLABROUS1 (Synthetic)Arabidopsis U6-26 promoterHygromycin Weber Weber Lab CRISPR plasmids (unpublished) Disruption of GLABROUS1 gene in Arabidopsis using CRISPR/Cas9 pUB-Cas9
    pTX168Hygromycin 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 with CaMV 35s promoter in plants pCAMBIA1300
  • Tag / Fusion Protein
    • SV40 NLS (C terminal on insert)
    		•
    pTX171Hygromycin 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 with CaMV 35s promoter in plants pTX168
  • Tag / Fusion Protein
    • SV40 NLS (C terminal on insert)
    		•
    pTX172Hygromycin 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 with Maize Ubiquitin1 promoter in plants pTX171
  • Tag / Fusion Protein
    • SV40 NLS (C terminal on insert)
    		•
    pTX176Hygromycin 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 estrodial-inducible STU (Single Transcriptional Unit) Cas9 in plants pTX171
  • Tag / Fusion Protein
    • SV40 NLS (C terminal on insert)
    		•
    pJG80TaCas9 (Other) Voytas High-efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR/Cas9. Plant J. 2016 Dec 10. doi: 10.1111/tpj.13446. Entry vector containing TaCas9-nos terminator between attL1 and attR5 sites unknown

    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.

    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)
    		•

    Activate

    A catalytically inactive Cas9 (dCas9) fused to a transcription activator peptide can increase transcription of a gene. Design your gRNA sequence to direct the dCas9-activator to a specific genomic sequence. Potential target locations can include promoter regions and regulatory regions. If the plasmid that you choose does not also express a gRNA, you will need to use a separate gRNA expression plasmid to target the dCas9-activator.

    Plasmid Gene/Insert Promoter Selectable Marker PI Publication Hidden Extra Search Info
    pBUN6A11dCas9-VP64 (Synthetic), gRNA scaffold (Synthetic)Ubi1p, OsU3pBar 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 dCas9-VP64, gRNA scaffold for insertion of target sequence (OsU3 promoter), Bar resistance pGreen-like binary vector
    pHSN6A01dCas9-VP64 (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 dCas9-VP64, gRNA scaffold for insertion of target sequence (AtU6-26 promoter), Hyg resistance pGreen-like binary vector
    pD10AH840AhCas9 (GB1041)Cas9 coding region with mutated (D10A, H840A) and inactivated catalytic domains (human codon optimised) (Other) Orzaez GoldenBraid 2.0: a comprehensive DNA assembly framework for plant synthetic biology. Plant Physiol. 2013 Jul;162(3):1618-31. Provides the human codon optimized CDS of Cas9 protein with mutated (D10A, H840A) and inactivated catalytic domains as a level 0 GoldenBraid part pUPD
    pYPQ152pco-dCas9-VP64 (Plant 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 pco-dCas9-VP64 pYPQ185-linker1
    pdCas9 (GB1079)Cas9 coding region with mutated (D10A, H840A) and inactivated catalytic domains (human codon optimised) (Other) Orzaez A modular toolbox for gRNA-Cas9 genome engineering in plants based on the GoldenBraid standard. Plant Methods. 2016 Feb 1;12:10. doi: 10.1186/s13007-016-0101-2. eCollection 2016. Provides the human codon optimized CDS of Cas9 protein with mutated (D10A, H840A) and inactivated catalytic domains as a level 0 GoldenBraid part for C-terminal fusions pUPD

    Intefere

    A catalytically inactive Cas9 (dCas9) or dCas9-repressor peptide fusion can be used to knock-down gene expression by interfering with transcription of the gene. Design your gRNA sequence to direct the dCas9 repressor to a specific genomic sequence. Potential target locations can include promoter regions, regulatory regions, and early coding regions. If the plasmid that you choose does not also express a gRNA, you will need to use a separate gRNA expression plasmid to target the dCas9 repressor.

    Plasmid Gene/Insert Promoter Selectable Marker PI Publication Hidden Extra Search Info
    pBUN6I11dCas9-KRAB (Synthetic), gRNA scaffold (Synthetic)Ubi1p, OsU3pBar 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 dCas9-KRAB, gRNA scaffold for insertion of target sequence (OsU3 promoter), Bar resistance pGreen-like binary vector
    pHSN6I01dCas9-KRAB (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 dCas9-KRAB, gRNA scaffold for insertion of target sequence (AtU6-26 promoter), Hyg resistance pGreen-like binary vector
    pEGB 35S:dCas9:Tnos (GB1191)dCas9 (Other)35S Orzaez GoldenBraid 2.0: a comprehensive DNA assembly framework for plant synthetic biology. Plant Physiol. 2013 Jul;162(3):1618-31. Transcriptional unit for human codon optimized with mutated (D10A, H840A) and inactivated catalytic domains Cas9 protein plant expression driven by the 35S promoter pDGB3alpha2
    pYPQ153pco-dCas9-3X(SRDX) (Plant 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 pco-dCas9-3X(SRDX) pYPQ185-linker1
    pdCas9 (GB1079)Cas9 coding region with mutated (D10A, H840A) and inactivated catalytic domains (human codon optimised) (Other) Orzaez A modular toolbox for gRNA-Cas9 genome engineering in plants based on the GoldenBraid standard. Plant Methods. 2016 Feb 1;12:10. doi: 10.1186/s13007-016-0101-2. eCollection 2016. Provides the human codon optimized CDS of Cas9 protein with mutated (D10A, H840A) and inactivated catalytic domains as a level 0 GoldenBraid part for C-terminal fusions pUPD
    pEGB 35s:dCas:BRD:tNos (GB1172)dCas9:BRD (Synthetic)35S Orzaez A modular toolbox for gRNA-Cas9 genome engineering in plants based on the GoldenBraid standard. Plant Methods. 2016 Feb 1;12:10. doi: 10.1186/s13007-016-0101-2. eCollection 2016. Transcriptional unit of (human codon optimized) inactivated Cas9 fused to the BRD Transcriptional Repressor pDGB3alpha2

    Empty gRNA Expression Vectors

    Select a gRNA expression plasmid based on factors such as selectable marker or cloning method. When using the CRISPR/Cas system, you will need to express both a Cas9 protein and a target-specific gRNA in the same cell at the same time. Single plasmids containing both the gRNA and Cas9 act as an all-in-one vector, but their function (cut, nick, activate, interfere...) is limited to that of the Cas9 present on the plasmid. gRNA plasmids that do not co-express Cas9 require a separate plasmid that does so; however, these independent gRNA plasmids can be paired with a wide variety of Cas9 plasmids and therefore are not limited to a single Cas9 function.

    gRNA Plasmid Promoter Cloning
    Enzyme(s)    		 Validated In Resistance Co-expressed Cas9 Depositing lab
    Cas9 species = S. pyogenes (PAM = NGG)
    pICSL01009::AtU6p aU6 BsaI Arabidopsis none, need Cas9 plasmid Kamoun
    Addgene Blugene icon

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