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CRISPR header icon CRISPR/Cas Plasmids for Use in Bacteria


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 bacteria.

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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 PI Publication Hidden Extra Search Info
pMJ806Cas9T7 Doudna A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science. 2012 Jun 28. pEC-K-MBP
pCas9tracr/Cas9 Marraffini RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol. 2013 Jan 29. doi: 10.1038/nbt.2508. Bacterial expression of Cas9 nuclease, tracrRNA and crRNA guide pACYC184
pwtCas9-bacteriawild-type Cas9pLtetO-1 Qi Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell. 2013 Feb 28;152(5):1173-83. doi: 10.1016/j.cell.2013.02.022. aTc-inducible expression of wild-type Cas9 (S .pyogenes) for bacterial gene knockdown pUC19
pET-28b-Cas9-HisCas9 (Other) Schier Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs. PLoS One. 2014 May 29;9(5):e98186. doi: 10.1371/journal.pone.0098186. eCollection 2014. For in vitro expression and purification of Cas9 protein pET-28b
DS-SPcasCas9 (Other), tracrRNA precursor (Other)proC, tracrRNA promoter Church Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat Methods. 2013 Sep 29. doi: 10.1038/nmeth.2681. Bacterial S. pyogenes Cas9 (SP) + tracrRNA expression, cloDF13/spectinomycin cloDF13-aadA
DS-NMcasCas9 (Other), NM tracrRNA (Other)proC, NM tracrRNA promoter Church Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat Methods. 2013 Sep 29. doi: 10.1038/nmeth.2681. Bacterial N. meningitidis Cas9 (NM) + tracrRNA expression, cloDF13/spectinomycin cloDF13-aadA
DS-ST1casCas9 (Other), ST1 tracrRNA (Other)proC Church Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat Methods. 2013 Sep 29. doi: 10.1038/nmeth.2681. Bacterial S. thermophilus #1 Cas9 (ST1) + tracrRNA expression, cloDF13/spectinomycin cloDF13-aadA
DS-TDcasCas9 (Other), TD tracrRNA (Other)proC Church Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat Methods. 2013 Sep 29. doi: 10.1038/nmeth.2681. Bacterial T. denticola Cas9 (TD) + tracrRNA expression, cloDF13/spectinomycin cloDF13-aadA
pET28a/Cas9-CysCas9-Cys (Other)T7 Promoter Kim Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Genome Res. 2014 Apr 2. Expresses N-terminal His tag fused human codon-optimized Cas9 nuclease having C-terminal Cysteine pET28a
pCRISPomyces-1sSpCas9 (Synthetic), tracrRNA (Other), crRNA cassette (Synthetic)rpsLp(XC)-BbsI, rpsLp(CF), gapdhp(EL) Zhao High-Efficiency Multiplex Genome Editing of Streptomyces Species Using an Engineered CRISPR/Cas System. ACS Synth Biol. 2014 Dec 8. Streptomyces expression of codon-optimized Cas9, tracrRNA, and custom crRNA pKC1139
pCRISPomyces-2sSpCas9 (Synthetic), gRNA cassette (Synthetic)rpsL(XC)-BbsI, gapdhp(EL) Zhao High-Efficiency Multiplex Genome Editing of Streptomyces Species Using an Engineered CRISPR/Cas System. ACS Synth Biol. 2014 Dec 8. Streptomyces expression of codon-optimized Cas9 and custom gRNA pKC1139
pCascas9 (Other)native cas9 promoter Yang Multigene editing in the Escherichia coli genome using the CRISPR-Cas9 system. Appl Environ Microbiol. 2015 Jan 30. pii: AEM.04023-14. Constitutive expression of cas9 and inducible expression of lambda RED and sgR pKD46
pET-(-30)dGFP-9xGGS-Cath-NLS-Cas9-6xHis(pos36)dGFP-9xGGS-Cath-NLS-Cas9-6xHis (Other)T7 Liu Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotechnol. 2015 Jan;33(1):73-80. doi: 10.1038/nbt.3081. Epub 2014 Oct 30. Expression of (pos36)dGFP-9xGGS-Cath-NLS-Cas9-6xHis in bacterial cells pET29
pET-Cas9-6xHisCas9_6xHis (Other)T7 Liu Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotechnol. 2015 Jan;33(1):73-80. doi: 10.1038/nbt.3081. Epub 2014 Oct 30. Expression of Cas9-6xHis in bacterial cells pET29
pTrex-b-NLS-hSpCas9NLS-hSpCas9Ribo-HX1 Tarleton CRISPR-Cas9-Mediated Single-Gene and Gene Family Disruption in Trypanosoma cruzi. MBio. 2014 Dec 30;6(1). pii: e02097-14. doi: 10.1128/mBio.02097-14. Expression of Cas9 in T. cruzi pTrex-b
pKCcas9dOScocas9 (Other), sgRNA (), act-orf4 homology armptipA, j23119 Jiang One-step high-efficiency CRISPR/Cas9-mediated genome editing in Streptomyces. Acta Biochim Biophys Sin (Shanghai). 2015 Apr;47(4):231-43. doi: 10.1093/abbs/gmv007. Epub 2015 Mar 3. High efficiency Streptomyces genome editing by CRISPR/Cas9 system pKC1139
pCas9-CR4Cas9 nuclease Prather The no-SCAR (Scarless Cas9 Assisted Recombineering) system for genome editing in Escherichia coli. Sci Rep. 2015 Oct 14;5:15096. doi: 10.1038/srep15096. Cas9 nuclease under control of pTet promoter with ssrA tag and constitutive tetR pdCas9-bacteria
SP-Cas9SP-CAS9 (Other)T7 Geijsen Efficient Intracellular Delivery of Native Proteins. Cell. 2015 Apr 23;161(3):674-690. doi: 10.1016/j.cell.2015.03.028. Expresses SP-Cas9 protein in bacterial cells pET-15
pCMKCas9 (Synthetic) Rodrigue Bacterial constitutive gRNA expression plasmid (unpublished) Bacterial constitutive S. pyogenes Cas9 expression plasmid pCMK
pET-Cas9-NLS-6xHisCas9-NLS-6xHis (Other)T7 Liu Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotechnol. 2015 Jan;33(1):73-80. doi: 10.1038/nbt.3081. Epub 2014 Oct 30. Expression of Cas9-NLS-6xHis in bacterial cells pET29
pET-NLS-Cas9-6xHisNLS-Cas9-6xHis (Other)T7 Liu Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotechnol. 2015 Jan;33(1):73-80. doi: 10.1038/nbt.3081. Epub 2014 Oct 30. Expression of NLS-Cas9-6xHis in bacterial cells pET29
pLPhygCAS9Humanized Streptococcus pyogenes Cas9 from PX330 (Addgene) (Other) Matlashewski CRISPR-Cas9-Mediated Genome Editing in Leishmania donovani. MBio. 2015 Jul 21;6(4). pii: e00861-15. doi: 10.1128/mBio.00861-15. Expresses CAS9 in Leishmania pLPHyg2
pYTK036Cas9 (Other) Dueber A Highly-characterized Yeast Toolkit for Modular, Multi-part Assembly. ACS Synth Biol. 2015 Apr 14. Encodes Cas9 as a Type 3 part to be used in the Dueber YTK system pYTK001
BPK764mammalian codon-optimized Streptococcus pyogenes Cas9-NLS-3XFlag, and SpCas9 gRNA (Other)T7 (x2) Joung Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature. 2015 Jun 22. doi: 10.1038/nature14592. Bacterial expression plasmid for SpCas9 & sgRNA (need to clone spacer into BsaI sites): T7-humanSpCas9-NLS-3xFLAG-T7-BsaIcassette-Sp-sgRNA pACYCDuet-1
  • Tags / Fusion Proteins
    • NLS (C terminal on insert)
    • 3x FLAG (C terminal on insert)
  • MSP1673mammalian codon-optimized Streptococcus thermophilus1 Cas9-NLS, and St1Cas9 gRNA (Other)T7 (x2) Joung Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature. 2015 Jun 22. doi: 10.1038/nature14592. Bacterial expression plasmid for S. thermophilus1 Cas9 & sgRNA (need to clone in spacer into BspMI sites): T7-humanSt1Cas9-NLS-T7-BspMIcassette-St1-sgRNA pACYCDuet-1
  • Tag / Fusion Protein
    • NLS (C terminal on insert)
  • BPK2101mammalian codon-optimized Staphylococcus aureus Cas9-NLS-3xFlag, and SaCas9 gRNA (Other)T7 (x2) Joung Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature. 2015 Jun 22. doi: 10.1038/nature14592. Bacterial expression plasmid for S. aureus Cas9 & sgRNA (need to clone in spacer into BsaI sites): T7-humanSaCas9-NLS-3xFLAG-T7-BsaIcassette-Sa-sgRNA pACYCDuet-1
  • Tags / Fusion Proteins
    • NLS (C terminal on insert)
    • 3x FLAG (C terminal on insert)
  • pHO4d-Cas9Cas9 (Other)T7 Nonet Landscape of target:guide homology effects on Cas9-mediated cleavage. Nucleic Acids Res. 2014 Dec 16;42(22):13778-87. doi: 10.1093/nar/gku1102. Epub 2014 Nov 15. Cas9nlsHis6 Bacterial Expression construct pHO4D
    pMJ915cas9 (Other)T7 Doudna Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. Elife. 2014 Dec 15;3:e04766. doi: 10.7554/eLife.04766. Express Streptococcus pyogenes Cas9 carrying two C-terminal SV40 NLS pML-2CT
    MSP2283mammalian codon-optimized Staphylococcus aureus Cas9-NLS-3xFlag (Other), SaCas9 sgRNA (+84)T7, T7 Joung Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition. Nat Biotechnol. 2015 Nov 2. doi: 10.1038/nbt.3404. Bacterial expression plasmid for SaCas9 & sgRNA targeted to site 1: T7-humanSaCas9-NLS-3xFLAG-T7-Sa-sgRNA(84) #1 pACYCDuet-1
    MSP2253mammalian codon-optimized KKH variant Staphylococcus aureus Cas9(E782K/N968K/R1015H)-NLS-3xFlag (Other), SaCas9 sgRNA (+84)T7, T7 Joung Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition. Nat Biotechnol. 2015 Nov 2. doi: 10.1038/nbt.3404. Bacterial expression plasmid for KKH SaCas9 & sgRNA targeted to site 1: T7-humanSaCas9(E782K/N968K/R1015H)-NLS-3xFLAG-T7-Sa-sgRNA(84) #1 pACYCDuet-1
    MSP2266mammalian codon-optimized Staphylococcus aureus Cas9-NLS-3xFlag (Other), SaCas9 sgRNA (+84)T7, T7 Joung Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition. Nat Biotechnol. 2015 Nov 2. doi: 10.1038/nbt.3404. Bacterial expression plasmid for SaCas9 & sgRNA targeted to site 2: T7-humanSaCas9-NLS-3xFLAG-T7-Sa-sgRNA(84) #2 pACYCDuet-1
    MSP2292mammalian codon-optimized KKH variant Staphylococcus aureus Cas9(E782K/N968K/R1015H)-NLS-3xFlag, and SaCas9 sgRNA(84) (Other), SaCas9 sgRNA (+84)T7, T7 Joung Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition. Nat Biotechnol. 2015 Nov 2. doi: 10.1038/nbt.3404. Bacterial expression plasmid for KKH SaCas9 & sgRNA targeted to site 2: T7-humanSaCas9(E782K/N968K/R1015H)-NLS-3xFLAG-T7-Sa-sgRNA(84) #2 pACYCDuet-1
    pCas9_sgRNA_0U6 promoter (Other), cas9 (Synthetic), tnos terminator (Other)U6 from Ustilago maydis, synthetic otef promoter (Spellig et al., 1996, Mol. Gen. Genet. 252) Kahmann Genome editing in Ustilago maydis using the CRISPR-Cas system. Fungal Genet Biol. 2015 Sep 11. pii: S1087-1845(15)30025-6. doi: 10.1016/j.fgb.2015.09.001. expresses Ustilago maydis codon-optimized Cas9, contains U. maydis U6 promoter, is self-replicating pNEBUC
    pMCSG7-Wt-NmeCas9pMCSG7-WT-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 Wildtype Nme cas9 with T7 promoter, N-terminal His tag and TEV site pMCSG7
    pCas9curCas9 (Other), Plasmid curing systempBAD Chen Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing. Metab Eng. 2015 Sep;31:13-21. doi: 10.1016/j.ymben.2015.06.006. Epub 2015 Jun 30. Constitutive expression of Cas9 gene and inducible expression of the plasmid curing system for CRISPR mediated genome editing of E. coli. pSC101ts
    pREDCas9Cas9 (Other), lambda red genes, plasmid curing systempBAD Chen Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing. Metab Eng. 2015 Sep;31:13-21. doi: 10.1016/j.ymben.2015.06.006. Epub 2015 Jun 30. Constitutive expression of Cas9, inducible expression of the Red recombineering system, and inducible expression of the plasmid curing system for CRISPR mediated genome editing of E. coli. pSC101ts
    pCAS1ylCas9 (Other), sgRNA (Synthetic)unknown Yang Multiplex gene editing of the Yarrowia lipolytica genome using the CRISPR-Cas9 system. J Ind Microbiol Biotechnol. 2016 Aug;43(8):1085-93. doi: 10.1007/s10295-016-1789-8. Epub 2016 Jun 27. Constitutive expression of Cas9 and sgRNA in Yarrowia lipolytica cells pMCSCen1
    pMA7CR_2.0cas9 (Other), lambda beta (Other), dam (Other), recX (Other)pTet, pAra, pAra, pTet Nielsen CRMAGE: CRISPR Optimized MAGE Recombineering. Sci Rep. 2016 Jan 22;6:19452. doi: 10.1038/srep19452. pMA7CR_2.0 contains arabinose inducible λ/RED β-protein and dam, and aTc inducible CRISPR/Cas9 and recX pBAD24
    pDEST-hisMBP-AsCpf1-ECAsCpf1 (Synthetic)tac promoter Kim Targeted mutagenesis in mice by electroporation of Cpf1 ribonucleoproteins. Nat Biotechnol. 2016 Jun 6. doi: 10.1038/nbt.3596. Expressing his-MBP tagged AsCpf1 (E.coli codon optimized) pDEST-hisMBP
    pMAL-his-LbCpf1-ECLbCpf1 (Synthetic)tac promoter Kim Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nat Biotechnol. 2016 Jun 6. doi: 10.1038/nbt.3609. Bacterial expression - MBP-his tagged LbCpf1 (E.coli codon optimized) pMAL-c5X
    pCas9-CATCas9 (Other), Chloramphenicol acetyltransferaseTgTUB1, TgTUB1 Lourido A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes. Cell. 2016 Sep 8;166(6):1423-1435.e12. doi: 10.1016/j.cell.2016.08.019. Epub 2016 Sep 2. Encodes Cas9 and chloramphenicol acetyltransferase (CAT) pU6-Universal
    pU6-DecoyDecoy sgRNAU6 Lourido A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes. Cell. 2016 Sep 8;166(6):1423-1435.e12. doi: 10.1016/j.cell.2016.08.019. Epub 2016 Sep 2. Encodes Cas9 and a CRISPR sgRNA that alleviates toxicity to Cas9 in Toxoplasma gondii pU6-Universal
    pEC-K-CBP_CjeCas9CjeCas9 (Other) Doudna Single-Stranded DNA Cleavage by Divergent CRISPR-Cas9 Enzymes. Mol Cell. 2015 Nov 5;60(3):398-407. doi: 10.1016/j.molcel.2015.10.030. Expresses Campylobacter jejuni Cas9 (Cje Cas9). pEC-K
    p2CT-CdiCdiCas9 Doudna Single-Stranded DNA Cleavage by Divergent CRISPR-Cas9 Enzymes. Mol Cell. 2015 Nov 5;60(3):398-407. doi: 10.1016/j.molcel.2015.10.030. Expresses Corynebacterium diphtheriae Cas9 (CdiCas9) PET
    pLdCNgRNA and Cas9 (Other)L. donovani ribosome RNA promoter Matlashewski Optimized CRISPR-Cas9 Genome Editing for Leishmania and Its Use To Target a Multigene Family, Induce Chromosomal Translocation, and Study DNA Break Repair Mechanisms. mSphere. 2017 Jan 18;2(1). pii: e00340-16. doi: 10.1128/mSphere.00340-16. eCollection 2017 Jan-Feb. Express gRNA and Cas9 in Leishmania with Neomycin resistance pSP72
    pLdCHgRNA and Cas9 (Other)L. donovani ribosome RNA promoter Matlashewski Optimized CRISPR-Cas9 Genome Editing for Leishmania and Its Use To Target a Multigene Family, Induce Chromosomal Translocation, and Study DNA Break Repair Mechanisms. mSphere. 2017 Jan 18;2(1). pii: e00340-16. doi: 10.1128/mSphere.00340-16. eCollection 2017 Jan-Feb. Express gRNA and Cas9 in Leishmania with Hygromycin resistance pSP72
    pJYS3_ΔcrtYfCpf1 (Other), crRNA of crtYf (Synthetic) Yang CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum. Nat Commun. 2017 May 4;8:15179. doi: 10.1038/ncomms15179. Constitutive transcription of FnCpf1 and crRNA of crtYf pXMJ19
    pJYS1PtacCpf1 (Other), recT (Other)tac, unknown Yang CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum. Nat Commun. 2017 May 4;8:15179. doi: 10.1038/ncomms15179. Constitutive trancription of FnCpf1 and recT in C.glutamitum, tac promoter pXMJ19
    pJYS1PeftuCpf1 (Other), recT (Other)etfu, unknown Yang CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum. Nat Commun. 2017 May 4;8:15179. doi: 10.1038/ncomms15179. Constitutive expression of FnCpf1 and recT in C.glutamitum, etfu promoter pXMJ19
    pX2-Cas9Cas9 (Other)pBAD Gill pX2-Cas9 (unpublished) Arabinose inducible Cas9 in a broad host range backbone. pBTBX-2
    pSHS212Cas9 (Other)T7 Doudna Conformational control of DNA target cleavage by CRISPR-Cas9. Nature. 2015 Nov 5;527(7576):110-3. doi: 10.1038/nature15544. Epub 2015 Oct 28. Cysteine-free (C80S/C574S) S. pyogenes Cas9 expression plasmid pCT10
    pSHS293Cas9 (Other)T7 Doudna Conformational control of DNA target cleavage by CRISPR-Cas9. Nature. 2015 Nov 5;527(7576):110-3. doi: 10.1038/nature15544. Epub 2015 Oct 28. D435C/E945C in cysteine-free (C80S/C574S) S. pyogenes Cas9 expression plasmid, lobe closure FRET construct pCT10
    pSHS306 - Bacterial expression plasmid for SpCas9, HNH FRET variantCas9 (Other)T7 Doudna Conformational control of DNA target cleavage by CRISPR-Cas9. Nature. 2015 Nov 5;527(7576):110-3. doi: 10.1038/nature15544. Epub 2015 Oct 28. S867C/S355C in cysteine-free (C80S/C574S) S. pyogenes Cas9 expression plasmid, HNH-1 FRET construct pCT10
    pSHS248Cas9 (Other)T7 Doudna Conformational control of DNA target cleavage by CRISPR-Cas9. Nature. 2015 Nov 5;527(7576):110-3. doi: 10.1038/nature15544. Epub 2015 Oct 28. S867C/N1054C in cysteine-free (C80S/C574S) S. pyogenes Cas9 expression plasmid, HNH-2 FRET construct pCT10
    pET-MBP-Geo_stGeoCas9 (Other) Doudna GeoCas9 Plasmids (unpublished) Expression plasmid for Cas9 from Geobacillus stearothermophilus with an N-Term MBP pET
    pET-MBP-NLS-Geo_stGeoCas9 (Other) Doudna GeoCas9 Plasmids (unpublished) Expression plasmid for Cas9 from Geobacillus stearothermophilus with an N-Term MBP and SV40 NLS pET
    pFC330Cas9 (Synthetic), pyrG (Other)Aspergillus nidulans tef1 promoter, native promoter Mortensen A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi. PLoS One. 2015 Jul 15;10(7):e0133085. doi: 10.1371/journal.pone.0133085. eCollection 2015. AMA1 plasmid with Aspergillus optimized Cas9 and pyrG selection marker custom
    pFC331Cas9 (Synthetic), argB (Other)Aspergillus nidulans tef1 promoter, native promoter Mortensen A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi. PLoS One. 2015 Jul 15;10(7):e0133085. doi: 10.1371/journal.pone.0133085. eCollection 2015. AMA1 plasmid with Aspergillus optimized Cas9 and argB selection marker custom
    pFC333Cas9 (Synthetic), ble (bleomycin resistance marker) (Other)Aspergillus nidulans tef1 promoter, Aspergillus nidulans trpC promoter Mortensen A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi. PLoS One. 2015 Jul 15;10(7):e0133085. doi: 10.1371/journal.pone.0133085. eCollection 2015. AMA1 plasmid with Aspergillus optimized Cas9 and ble selection marker custom
    pFC332Cas9 (Synthetic), hph (hygromycin resistance marker) (Other)Aspergillus nidulans tef1 promoter, Aspergillus nidulans trpC promoter Mortensen A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi. PLoS One. 2015 Jul 15;10(7):e0133085. doi: 10.1371/journal.pone.0133085. eCollection 2015. AMA1 plasmid with Aspergillus optimized Cas9 and hph selection marker custom
    pMJ915v2+Nterm Spe1 +Cterm Age1 site Doudna Efficient genome editing in the mouse brain by local delivery of engineered Cas9 ribonucleoprotein complexes. Nat Biotechnol. 2017 Feb 13. doi: 10.1038/nbt.3806. For expression of modified SpyCas9 in e.coli. pMJ915
    1xNLS-pMJ915v2Cas9 Doudna Efficient genome editing in the mouse brain by local delivery of engineered Cas9 ribonucleoprotein complexes. Nat Biotechnol. 2017 Feb 13. doi: 10.1038/nbt.3806. For expression of modified SpyCas9 in e.coli. pMJ915
    2xNLS-pMJ915v2Cas9, T7 Doudna Efficient genome editing in the mouse brain by local delivery of engineered Cas9 ribonucleoprotein complexes. Nat Biotechnol. 2017 Feb 13. doi: 10.1038/nbt.3806. For expression of modified SpyCas9 in e.coli. pMJ915
    4xNLS-pMJ915v2Cas9, T7 Doudna Efficient genome editing in the mouse brain by local delivery of engineered Cas9 ribonucleoprotein complexes. Nat Biotechnol. 2017 Feb 13. doi: 10.1038/nbt.3806. For expression of modified SpyCas9 in e.coli. pMJ915
    pMJ915v2 + sfGFPCas9, T7 Doudna Efficient genome editing in the mouse brain by local delivery of engineered Cas9 ribonucleoprotein complexes. Nat Biotechnol. 2017 Feb 13. doi: 10.1038/nbt.3806. For expression of modified SpyCas9 in e.coli. pMJ915
    1xNLS-pMJ915v2-sfGFPCas9, T7 Doudna Efficient genome editing in the mouse brain by local delivery of engineered Cas9 ribonucleoprotein complexes. Nat Biotechnol. 2017 Feb 13. doi: 10.1038/nbt.3806. For expression of modified SpyCas9 in e.coli. pMJ915
    2xNLS-pMJ915v2-sfGFPCas9, T7 Doudna Efficient genome editing in the mouse brain by local delivery of engineered Cas9 ribonucleoprotein complexes. Nat Biotechnol. 2017 Feb 13. doi: 10.1038/nbt.3806. For expression of modified SpyCas9 in e.coli. pMJ915
    4xNLS-pMJ915v2-sfGFPCas9, T7 Doudna Efficient genome editing in the mouse brain by local delivery of engineered Cas9 ribonucleoprotein complexes. Nat Biotechnol. 2017 Feb 13. doi: 10.1038/nbt.3806. For expression of modified SpyCas9 in e.coli. pMJ915
    7xNLS-pMJ915v2-sfGFPCas9, T7 Doudna Efficient genome editing in the mouse brain by local delivery of engineered Cas9 ribonucleoprotein complexes. Nat Biotechnol. 2017 Feb 13. doi: 10.1038/nbt.3806. For expression of modified SpyCas9 in e.coli. pMJ915
    pET-CjCas9CjCas9 (Other)T7 Kim In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni. Nat Commun. 2017 Feb 21;8:14500. doi: 10.1038/ncomms14500. Expression of CjCas9 with His tag in E.coli pET28b
    pEM-Cas9HF1Cas9HF1 (Other)pTet Lynch Lynch Lab CRISPR/Cas9 plasmids (unpublished) Modified from pCas9-CR4 (Addgene: 62655) to use the high-fidelity version of Cas9, SpCas9-HF1 (N497A/R661A/Q695A/Q926A) from Kleinstiver et al 2016. pCas9-CR4
    pEM-Cas9HF1-recA56Cas9HF1 (Other), recA56 (Other)pTet, proD Lynch Lynch Lab CRISPR/Cas9 plasmids (unpublished) Modified from pEM-Cas9HF1 (Addgene ID: 89961) to include constitutive recA56 to block recA-mediated double-strand break repair. pEM-Cas9HF1
    6-His-MBP-TEV-FnCpf1FnCpf1 (humanized) (Other)T7 Zhang Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell. 2015 Sep 23. pii: S0092-8674(15)01200-3. doi: 10.1016/j.cell.2015.09.038. Bacterial expression plasmid for protein purification pET-28
    6His-MBP-TEV-huAsCpf1huAsCpf1 (Other) Zhang Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array. Nat Biotechnol. 2017 Jan;35(1):31-34. doi: 10.1038/nbt.3737. Epub 2016 Dec 5. Bacterial expression plasmid for protein purification pET-28
    6His-MBP-TEV-huLbCpf1huLbCpf1 (Other) Zhang Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array. Nat Biotechnol. 2017 Jan;35(1):31-34. doi: 10.1038/nbt.3737. Epub 2016 Dec 5. Bacterial expression plasmid for protein purification pET-28
    pMST665_BB3_L 23_gRNAempty_cas9_BbsICas9 Sauer An efficient tool for metabolic pathway construction and gene integration for Aspergillus niger. Bioresour Technol. 2017 May 4. pii: S0960-8524(17)30643-0. doi: 10.1016/j.biortech.2017.05.004. BB3_L 23_gRNAempty_cas9_BbsI; CRISPR plasmid with empty gRNA spacer to incorporate gRNA, Cas9 BB3_AMA_2.8_pUC-ORI_L_AC_hph
    pET28a-Cas9-HisNLS-Cas9-NLS (Other)T7 Gao Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes. Nat Commun. 2017 Jan 18;8:14261. doi: 10.1038/ncomms14261. Purification of Cas9 protein pET28a (+)
    pJWV102-wtcas9spcas9sp (Synthetic)PczcD Kjos Chromosome segregation drives division site selection in Streptococcus pneumoniae. Proc Natl Acad Sci U S A. 2017 Jul 3. pii: 201620608. doi: 10.1073/pnas.1620608114. Plasmid containing wild-type cas9sp pJWV102
    pThermoCas9_ctrlCas9 from the type IIc CRISPR-Cas sytem of Geobacillus thermodenitrificans T12 strain (Other), ThermoCas9 single guide RNA expressing module (Other)B. smithii xylL promoter, B. coagulans DSM 1 pta promoter van der Oost Characterizing a thermostable Cas9 for bacterial genome editing and silencing bioRxiv (2017) 177717 Expresses ThermoCas9 and its sgRNA module pNW33n
    SpyCas9(WT)SpyCas9 (Synthetic) Huang Structural basis of CRISPR-SpyCas9 inhibition by an anti-CRISPR protein. Nature. 2017 Jun 15;546(7658):436-439. doi: 10.1038/nature22377. Epub 2017 Apr 27. Express Streptococcus pyogenes Cas9 pGEX-6P-1
  • Tag / Fusion Protein
    • GST (N terminal on backbone)
  • Interfere

    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 PI Publication Hidden Extra Search Info
    pMJ841Cas9 (Other)T7 Doudna A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science. 2012 Jun 28. pEC-K-MBP
  • Tags / Fusion Proteins
    • MBP (N terminal on backbone)
    • His6 (N terminal on backbone)
  • pdCas9-bacteriadCas9 (bacteria) (Other)pLtetO-1 Qi Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell. 2013 Feb 28;152(5):1173-83. doi: 10.1016/j.cell.2013.02.022. aTc-inducible expression of a catalytically inactive bacterial Cas9 (S. pyogenes) for bacterial gene knockdown p15A vector
    pdCas9tracrRNA (Other), dcas9 (Other), CRISPR array Marraffini Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res. 2013 Jun 12. Expresses the tracrRNA, the dCas9 catalytic site mutant and a CRISPR array designed for the easy cloning of new spacers. pACYC184
    DS-SPcasN-Cas9, nuclease-null (Other), tracrRNA precursor (Other)proC, tracdrRNA promoter Church Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat Methods. 2013 Sep 29. doi: 10.1038/nmeth.2681. Bacterial nuclease-null SP Cas9 expression cloDF13-aadA
    10xHis-MBP-TEV-S. pyogenes dCas9 M1C D10A C80S H840A C574SdCas9 M1C D10A C80S H840A C574S (Other) Doudna Programmable RNA recognition and cleavage by CRISPR/Cas9. Nature. 2014 Sep 28. doi: 10.1038/nature13769. dCas9 with single cysteine residue (M1C) for site-specific labelling pHMGWA
    pAN-PTet-dCas9dCas9 (Other)Ptet Voigt Multi-input CRISPR/Cas genetic circuits that interface host regulatory networks. Mol Syst Biol. 2014 Nov 24;10:763. doi: 10.15252/msb.20145735. controls the expression of S. pyogenes dCas9 from a Tc‐inducible PTet promoter. unknown
    pCRISPathBrickConstitutive native promoters Koffas CRISPathBrick: Modular Combinatorial Assembly of Type II-A CRISPR Arrays for dCas9-Mediated Multiplex Transcriptional Repression in E. coli. ACS Synth Biol. 2015 Mar 30. E. coli vector for expression of S. pyogenes dCas9, tracrRNA, and nontargeting CRISPR array with BsaI site for inserting user-defined spacer-repeat bricks pdCas9-Marraffini (pACYC184)
    MSP712mammalian codon-optimized Streptococcus pyogenes dCas9 (D10A/H840A)-NLS-3XFlag, and SpCas9 gRNA (Other)T7 (x2) Joung Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature. 2015 Jun 22. doi: 10.1038/nature14592. Bacterial expression plasmid for Sp-dCas9 & sgRNA (need to clone in spacer into BsaI sites): T7-humanSpdCas9(D10A/H840A)-T7-BsaIcassette-Sp-sgRNA pACYCDuet-1
  • Tags / Fusion Proteins
    • NLS (C terminal on insert)
    • 3x FLAG (C terminal on insert)
  • pMM704dCas9, LacI, sgRNA Lu Programming a Human Commensal Bacterium, Bacteroides thetaiotaomicron, to Sense and Respond to Stimuli in the Murine Gut Microbiota Cell Systems, 2015 IPTG-inducible CRISPRi vector targeting BT1854, pNBU2 backbone, AmpR pExchange-tdk
    pET302-6His-dCas9-HaloHis6-dCas9-Halo (Synthetic) Lionnet CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells. Proc Natl Acad Sci U S A. 2015 Sep 22;112(38):11870-5. doi: 10.1073/pnas.1515692112. Epub 2015 Aug 31. Encodes a Halo-labeled fusion of nuclease deficient Cas9 pET302
    pMD19T-psba1-Ppsba2-dCas9-SpRdCas9 from S. pyogenes (Other)PpsbA2 Hudson Multiple Gene Repression in Cyanobacteria Using CRISPRi. ACS Synth Biol. 2015 Dec 28. Contains dCas9 from S. pyogenes under constitutive promoter. Suicide vector inserts into psba1 site of Synechocystis. Carries spectinomycin resist. Recommend E. coli Copy cutter for propogation. pMD19T simple
    pMD19T-psba1-TetR-PL22-dCas9-SpRdCas9 from S. pyogenes (Other)PL22 Hudson Multiple Gene Repression in Cyanobacteria Using CRISPRi. ACS Synth Biol. 2015 Dec 28. Contains dCas9 from S. pyogenes under aTc inducible promoter. Suicide vector inserts into psba1 site of Synechocystis. Carries spectinomycin resist. Recommend E. coli Copy cutter for propogation. pMD19T simple
    pdCas9-M-C4Repressor C4 (orthogonal T7-lac repressor) (Synthetic)Constitutive wild-type S. pyogenes promoter Koffas Rapid generation of CRISPR/dCas9-regulated, orthogonally repressible hybrid T7-lac promoters for modular, tuneable control of metabolic pathway fluxes in Escherichia coli. Nucleic Acids Res. 2016 Apr 13. pii: gkw231. CRISPR synthetic transcription factor repressor plasmid encoding dCas9, tracrRNA, and a single spacer CRISPR array encoding crRNA for orthogonal repression of T7-lac promoter variant C4. pdCas9
    pdCas9-M-3F2Repressor C4 (orthogonal T7-lac repressor) (Synthetic)Constitutive wild-type S. pyogenes promoter Koffas Rapid generation of CRISPR/dCas9-regulated, orthogonally repressible hybrid T7-lac promoters for modular, tuneable control of metabolic pathway fluxes in Escherichia coli. Nucleic Acids Res. 2016 Apr 13. pii: gkw231. CRISPR synthetic transcription factor repressor plasmid encoding dCas9, tracrRNA, and a single spacer CRISPR array encoding crRNA for orthogonal repression of T7-lac promoter variant 3F2. pdCas9
    pdCas9-M-3H5Repressor 3H5 (orthogonal T7-lac repressor) (Synthetic)Constitutive wild-type S. pyogenes promoter Koffas Rapid generation of CRISPR/dCas9-regulated, orthogonally repressible hybrid T7-lac promoters for modular, tuneable control of metabolic pathway fluxes in Escherichia coli. Nucleic Acids Res. 2016 Apr 13. pii: gkw231. CRISPR synthetic transcription factor repressor plasmid encoding dCas9, tracrRNA, and a single spacer CRISPR array encoding crRNA for orthogonal repression of T7-lac promoter variant 3H5. pdCas9
    pdCas9-M-1B6Repressor 1B6 (orthogonal T7-lac repressor) (Synthetic)Constitutive wild-type S. pyogenes promoter Koffas Rapid generation of CRISPR/dCas9-regulated, orthogonally repressible hybrid T7-lac promoters for modular, tuneable control of metabolic pathway fluxes in Escherichia coli. Nucleic Acids Res. 2016 Apr 13. pii: gkw231. CRISPR synthetic transcription factor repressor plasmid encoding dCas9, tracrRNA, and a single spacer CRISPR array encoding crRNA for orthogonal repression of T7-lac promoter variant 1B6. pdCas9
    pdCas9-M-4F2Repressor 4F2 (orthogonal T7-lac repressor) (Synthetic)Constitutive wild-type S. pyogenes promoter Koffas Rapid generation of CRISPR/dCas9-regulated, orthogonally repressible hybrid T7-lac promoters for modular, tuneable control of metabolic pathway fluxes in Escherichia coli. Nucleic Acids Res. 2016 Apr 13. pii: gkw231. CRISPR synthetic transcription factor repressor plasmid encoding dCas9, tracrRNA, and a single spacer CRISPR array encoding crRNA for orthogonal repression of T7-lac promoter variant 4F2. pdCas9
    pdCas9-M-5F5Repressor 5F5 (orthogonal T7-lac repressor) (Synthetic)Constitutive wild-type S. pyogenes promoter Koffas Rapid generation of CRISPR/dCas9-regulated, orthogonally repressible hybrid T7-lac promoters for modular, tuneable control of metabolic pathway fluxes in Escherichia coli. Nucleic Acids Res. 2016 Apr 13. pii: gkw231. CRISPR synthetic transcription factor repressor plasmid encoding dCas9, tracrRNA, and a single spacer CRISPR array encoding crRNA for orthogonal repression of T7-lac promoter variant 5F5. pdCas9
    pdCas9-M-1D4Repressor 1D4 (orthogonal T7-lac repressor) (Synthetic)Constitutive wild-type S. pyogenes promoter Koffas Rapid generation of CRISPR/dCas9-regulated, orthogonally repressible hybrid T7-lac promoters for modular, tuneable control of metabolic pathway fluxes in Escherichia coli. Nucleic Acids Res. 2016 Apr 13. pii: gkw231. CRISPR synthetic transcription factor repressor plasmid encoding dCas9, tracrRNA, and a single spacer CRISPR array encoding crRNA for orthogonal repression of T7-lac promoter variant 1D4. pdCas9
    pdCas9-M-4A6Repressor 4A6 (orthogonal T7-lac repressor) (Synthetic)Constitutive wild-type S. pyogenes promoter Koffas Rapid generation of CRISPR/dCas9-regulated, orthogonally repressible hybrid T7-lac promoters for modular, tuneable control of metabolic pathway fluxes in Escherichia coli. Nucleic Acids Res. 2016 Apr 13. pii: gkw231. CRISPR synthetic transcription factor repressor plasmid encoding dCas9, tracrRNA, and a single spacer CRISPR array encoding crRNA for orthogonal repression of T7-lac promoter variant 4A6. pdCas9
    pdCas9-M-3A2Repressor 3A2 (orthogonal T7-lac repressor) (Synthetic)Constitutive wild-type S. pyogenes promoter Koffas Rapid generation of CRISPR/dCas9-regulated, orthogonally repressible hybrid T7-lac promoters for modular, tuneable control of metabolic pathway fluxes in Escherichia coli. Nucleic Acids Res. 2016 Apr 13. pii: gkw231. CRISPR synthetic transcription factor repressor plasmid encoding dCas9, tracrRNA, and a single spacer CRISPR array encoding crRNA for orthogonal repression of T7-lac promoter variant 3A2. pdCas9
    pdCas9-M-1E4Repressor 1E4 (orthogonal T7-lac repressor) (Synthetic)Constitutive wild-type S. pyogenes promoter Koffas Rapid generation of CRISPR/dCas9-regulated, orthogonally repressible hybrid T7-lac promoters for modular, tuneable control of metabolic pathway fluxes in Escherichia coli. Nucleic Acids Res. 2016 Apr 13. pii: gkw231. CRISPR synthetic transcription factor repressor plasmid encoding dCas9, tracrRNA, and a single spacer CRISPR array encoding crRNA for orthogonal repression of T7-lac promoter variant 1E4. pdCas9
    pdCas9-M-G6Repressor G6 (orthogonal T7-lac repressor) (Synthetic)Constitutive wild-type S. pyogenes promoter Koffas Rapid generation of CRISPR/dCas9-regulated, orthogonally repressible hybrid T7-lac promoters for modular, tuneable control of metabolic pathway fluxes in Escherichia coli. Nucleic Acids Res. 2016 Apr 13. pii: gkw231. CRISPR synthetic transcription factor repressor plasmid encoding dCas9, tracrRNA, and a single spacer CRISPR array encoding crRNA for orthogonal repression of T7-lac promoter variant G6. pdCas9
    pdCASclosdCas9 (Other), sgRNA to spo0Aptb, Pj23119 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. Transcriptional repression for gene Spo0A (CAC-2011) in Clostridium acetobutylicum ATCC 824 pIMP1-ptb
    pZ8-T_dCas9dcas9ptac Lu Corynebacterium glutamicum Metabolic Engineering with CRISPR Interference (CRISPRi). ACS Synth Biol. 2016 Feb 16. pZ8-1 plasmid carrying dcas9, driven by the IPTG-inducible Ptac promoter, KanR pZ8-1
    pZ8-P_dCas9dcas9Propionate inducible promoter (prp) Lu Corynebacterium glutamicum Metabolic Engineering with CRISPR Interference (CRISPRi). ACS Synth Biol. 2016 Feb 16. pZ8-1 plasmid carrying dcas9 driven by the propionate-inducible prpD2 promoter (PprpD2), KanR pZ8-Prp
    pJMP1dCas9 (Other)xylA Gross A Comprehensive, CRISPR-based Functional Analysis of Essential Genes in Bacteria. Cell. 2016 Jun 2;165(6):1493-506. doi: 10.1016/j.cell.2016.05.003. Epub 2016 May 26. Bacillus subtilis dCas9 expression vector; integrates into lacA/ganA pAX01
    pCas2AdCas9PEZ3 Pfleger CRISPR interference as a titratable, trans-acting regulatory tool for metabolic engineering in the cyanobacterium Synechococcus sp. strain PCC 7002. Metab Eng. 2016 Jul 29. pii: S1096-7176(16)30062-3. doi: 10.1016/j.ymben.2016.07.007. dCas9 expressed from aTc-inducible promoter in acsA, A RBS: AGGAGA pACSA
    pCas2BdCas9PEZ3 Pfleger CRISPR interference as a titratable, trans-acting regulatory tool for metabolic engineering in the cyanobacterium Synechococcus sp. strain PCC 7002. Metab Eng. 2016 Jul 29. pii: S1096-7176(16)30062-3. doi: 10.1016/j.ymben.2016.07.007. dCas9 expressed from aTc-inducible promoter in acsA, B RBS: TCGAGA pACSA
    pCas2CdCas9PEZ3 Pfleger CRISPR interference as a titratable, trans-acting regulatory tool for metabolic engineering in the cyanobacterium Synechococcus sp. strain PCC 7002. Metab Eng. 2016 Jul 29. pii: S1096-7176(16)30062-3. doi: 10.1016/j.ymben.2016.07.007. dCas9 expressed from aTc-inducible promoter in acsA, C RBS: TGGACA pACSA
    pCas2DdCas9PEZ3 Pfleger CRISPR interference as a titratable, trans-acting regulatory tool for metabolic engineering in the cyanobacterium Synechococcus sp. strain PCC 7002. Metab Eng. 2016 Jul 29. pii: S1096-7176(16)30062-3. doi: 10.1016/j.ymben.2016.07.007. dCas9 expressed from aTc-inducible promoter in acsA, D RBS: AGGACG pACSA
    pCas2EdCas9PEZ3 Pfleger CRISPR interference as a titratable, trans-acting regulatory tool for metabolic engineering in the cyanobacterium Synechococcus sp. strain PCC 7002. Metab Eng. 2016 Jul 29. pii: S1096-7176(16)30062-3. doi: 10.1016/j.ymben.2016.07.007. dCas9 expressed from aTc-inducible promoter in acsA, E RBS: AGGGCG pACSA
    pCas2FdCas9PEZ3 Pfleger CRISPR interference as a titratable, trans-acting regulatory tool for metabolic engineering in the cyanobacterium Synechococcus sp. strain PCC 7002. Metab Eng. 2016 Jul 29. pii: S1096-7176(16)30062-3. doi: 10.1016/j.ymben.2016.07.007. dCas9 expressed from aTc-inducible promoter in acsA, F RBS: TGGGCG pACSA
    pCas7dCas9c225 Pfleger CRISPR interference as a titratable, trans-acting regulatory tool for metabolic engineering in the cyanobacterium Synechococcus sp. strain PCC 7002. Metab Eng. 2016 Jul 29. pii: S1096-7176(16)30062-3. doi: 10.1016/j.ymben.2016.07.007. dCas9 expressed from low constituve promoter, c225, in acsA pACSA
    pCas8dCas9none Pfleger CRISPR interference as a titratable, trans-acting regulatory tool for metabolic engineering in the cyanobacterium Synechococcus sp. strain PCC 7002. Metab Eng. 2016 Jul 29. pii: S1096-7176(16)30062-3. doi: 10.1016/j.ymben.2016.07.007. dCas9 without a promoter in acsA pACSA
    pRH2502dcas9 (Other)uv15tetO Husson Investigating essential gene function in Mycobacterium tuberculosis using an efficient CRISPR interference system. Nucleic Acids Res. 2016 Jul 12. pii: gkw625. Expression of dcas9 D10A H840A from a TetR-regulated uvtetO promoter pTC-0X-1L
    pAW019-2dcas9 (Other)PxylA (B. megaterium) Chou Development of a CRISPR-Cas9 toolkit for comprehensive engineering of Bacillus subtilis. Appl Environ Microbiol. 2016 Jun 3. pii: AEM.01159-16. Inducible dCas9 integration vector for Bacillus subtilis pAX01 (ColE1)
    Jun lab tunable CRISPRi strain Jun tCRISPRi: tunable and reversible, one-step control of gene expression. Sci Rep. 2016 Dec 20;6:39076. doi: 10.1038/srep39076. Strain SJ_XTL219 Genotype: MG1655 PBAD_dcas9 ΔlacI ΔaraE araFGH<>spec lacY A177C, galM<pBBa-J23119-tet-sacB-handle-term strain: SJ_XTL219

    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 PI Publication Hidden Extra Search Info
    pWJ66tracrRNA (Other), dcas9-w (Other), CRISPR array Marraffini Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res. 2013 Jun 12. Same as pdCas9, but with the dCas9-w fusion (w is fused at the C-terminal end of dCas9) pACYC184
    pWJ68tracrRNA (Other), w-dcas9 (Other), CRISPR array Marraffini Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res. 2013 Jun 12. Same as pdCas9, but with the w-dCas9 fusion (w is fused at the N-terminal end of dCas9) pACYC184
    pET-dCas9-VP64-6xHisdCas9-VP64 (Other)T7 Liu Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotechnol. 2015 Jan;33(1):73-80. doi: 10.1038/nbt.3081. Epub 2014 Oct 30. Expression of dCas9-VP64-6xHis in bacterial cells pET29
    pET-deSpCas9-VP64-6xHisdead/inactive eSpCas9-NLS-3xFLAG-VP64 (Other)T7 Welker Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage. Genome Biol. 2017 Oct 6;18(1):190. doi: 10.1186/s13059-017-1318-8. Expression of dead/inactive increased fidelity eSpCas9 (1.1)-VP64-6xHis in bacterial cells pET29
    pET-dSpCas9-HF1-VP64-6xHisdead/inactive SpCas9-HF1-NLS-3xFLAG-VP64 (Other)T7 Welker Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage. Genome Biol. 2017 Oct 6;18(1):190. doi: 10.1186/s13059-017-1318-8. Expression of dead/inactive increased fidelity SpCas9-HF1-VP64-6xHis in bacterial cells pET29
    pET-dHeFSpCas9-VP64-6xHisdead/inactive HeFSpCas9-NLS-3xFLAG-VP64 (Other)T7 Welker Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage. Genome Biol. 2017 Oct 6;18(1):190. doi: 10.1186/s13059-017-1318-8. Expression of dead/inactive increased fidelity HeFSpCas9-VP64-6xHis in bacterial cells pET29

    Purify

    A catalytically inactive Cas9 (dCas9) fused to an epitope tag(s) can be used to purify genomic DNA bound by the gRNA. Design your gRNA sequence to direct the dCas9 to a specific genomic sequence. 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.

    Plasmid Gene/Insert Promoter PI Publication
    3xFLAG-dCas9/p-bacteria 3xFLAG-dCas9 pLtetO-1 Fujii Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR. Biochem Biophys Res Commun. 2013 Aug 11. pii: S0006-291X(13)01329-6. doi: 10.1016/j.bbrc.2013.08.013.

    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)
    pCRISPR BsaI E. coli,
    S. pneumoniae
    Kanamycin none, need Cas9 plasmid Marraffini
    pCas9 BsaI E. coli,
    S. pneumoniae
    Chloramphenicol yes, cut Marraffini
    pgRNA-bacteria BBa_J23119 SpeI + HindIII Ampicillin none, need Cas9 plasmid Qi
    Addgene Blugene icon

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