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CRISPR header icon CRISPR/Cas9 plasmids for use in Yeast


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 yeast.

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
p414-TEF1p-Cas9-CYC1tHuman Optimized S. pyogenes Cas9 (Homo sapiens)TEF1 promoterTRP1 Church Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res p414
p415-GalL-Cas9-CYC1tHuman Optimized S. pyogenes Cas9 (Homo sapiens)GalLLEU2 Church Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res p415
pACT2-CAS9cCas9c (Synthetic)yeast ADH1 promoterLEU2 Zhao Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing. J Integr Plant Biol. 2013 Dec 30. doi: 10.1111/jipb.12152. Express Eukaryotic-codon-optimized Cas9c gene in yeast under ADH1 promoter for genome editing. SV40 NLS is fused to the C terminal. GAL4 region from pACT2 is removed. pACT2
pMZ222Cas9 (Other)adh1LEU2 Zaratiegui Implementation of the CRISPR-Cas9 system in fission yeast. Nat Commun. 2014 Oct 29;5:5344. doi: 10.1038/ncomms6344. Constitutive expression of humanized Cas9 pART1
pMZ374Cas9 (Other), rrk1:sgRNA (Other)adh1, rrk1ura4 Zaratiegui Implementation of the CRISPR-Cas9 system in fission yeast. Nat Commun. 2014 Oct 29;5:5344. doi: 10.1038/ncomms6344. Combination adh1:cas9/rrk1:sgRNA for CRISPR genome editing in fission yeast: Empty sgRNA target (CspCI placeholder) (see comments). pMZ283
pCTiCas9 (Other), tracrRNA (Other)TEF1p, RPR1pLEU2 Zhao Homology-Integrated CRISPR-Cas (HI-CRISPR) System for One-Step Multigene Disruption in Saccharomyces cerevisiae. ACS Synth Biol. 2014 Sep 19. Plasmid encoding iCas9 and tracrRNA on pRS415 backbone pRS415
pCRCTiCas9 (Other), tracrRNA (Other)TEF1p, RPR1pURA3 Zhao Homology-Integrated CRISPR-Cas (HI-CRISPR) System for One-Step Multigene Disruption in Saccharomyces cerevisiae. ACS Synth Biol. 2014 Sep 19. Plasmid encoding iCas9, tracrRNA and crRNAs pRS426
Cas9-NATHuman-optimized S. pyogenes Cas9 with Clonnat marker gene expression cassetteNourseothricin(clonNat) Jin Construction of a quadruple auxotrophic mutant of an industrial polyploid saccharomyces cerevisiae strain by using RNA-guided Cas9 nuclease. Appl Environ Microbiol. 2014 Dec;80(24):7694-701. doi: 10.1128/AEM.02310-14. Epub 2014 Oct 3. Cas9 expression cassette carried by a yeast single-copy episome plasmid pRS414-TEF1p-Cas9-CYC1t
pML104Cas9 (Saccharomyces cerevisiae), single guide RNA expression cassette (Saccharomyces cerevisiae)pTDH3, pSNR52URA3 Wyrick New vectors for simple and streamlined CRISPR-Cas9 genome editing in Saccharomyces cerevisiae. Yeast. 2015 Dec;32(12):711-20. doi: 10.1002/yea.3098. Epub 2015 Sep 21. Expresses Cas9 and contains guide RNA expression cassette with BclI-SwaI cloning sites for guide sequence cloning; Contains URA3 marker for yeast transformation pRSII426
pML107Cas9 (Saccharomyces cerevisiae), single guide RNA expression cassette (Saccharomyces cerevisiae)pTDH3, pSNR52LEU2 Wyrick New vectors for simple and streamlined CRISPR-Cas9 genome editing in Saccharomyces cerevisiae. Yeast. 2015 Dec;32(12):711-20. doi: 10.1002/yea.3098. Epub 2015 Sep 21. Expresses Cas9 and contains guide RNA expression cassette with BclI-SwaI cloning sites for guide sequence cloning; Contains LEU2 marker for yeast transformation pRSII425
pJH001Cas9 (Saccharomyces cerevisiae), EmptyLEU2 Wyrick New vectors for simple and streamlined CRISPR-Cas9 genome editing in Saccharomyces cerevisiae. Yeast. 2015 Dec;32(12):711-20. doi: 10.1002/yea.3098. Epub 2015 Sep 21. High copy Cas9 expression vector with LEU2 marker for yeast transformation pRSII425
p415-PtrpC-Cas9-TtrpC-CYC1thumanized Cas9 (Homo sapiens)LEU2 Hong Efficient gene editing in Neurospora crassa with CRISPR technology Fungal Biology and Biotechnology 2015, 2:4 Express humanized Cas9 under the control of trpC promoter and terminator from A. nidulans. pRS415
pCRISPRylCodon optimized Cas9 from S. pyogenes (Synthetic), sgRNA expression cassette (Synthetic)UAS1B8-TEF(136), SCR1'-tRNALEU2 Wheeldon Synthetic RNA polymerase III promoters facilitate high efficiency CRISPR-Cas9 mediated genome editing in Yarrowia lipolytica. ACS Synth Biol. 2015 Dec 29. CRISPR/Cas9 vector for Yarrowia lipolytica, with AvrII site for sgRNA insertion pUC19
pMZ376Cas9 (Other), rrk1:sgRNA (Other)adh1, rrk1Neomycin (select with G418) Zaratiegui sgRNA/Cas9 (unpublished) Combined sgRNA/Cas9 vector with KanMX marker pMZ374
pMZ377Cas9 (Other), rrk1:sgRNA (Other)adh1, rrk1LEU2 Zaratiegui sgRNA/Cas9 (unpublished) Combined sgRNA/Cas9 vector with LEU2 marker pMZ374
pMZ379Cas9 (Other), rrk1:sgRNA (Other)adh1, rrk1nourseothricin Zaratiegui sgRNA/Cas9 (unpublished) Combined sgRNA/Cas9 vector with NatMX marker pMZ374
pCfB2312 (TEF1p-Cas9-CYC1t_kanMX)Cas9 (Other)Gentamicin Borodina EasyClone-MarkerFree: A vector toolkit for marker-less integration of genes into Saccharomyces cerevisiae via CRISPR-Cas9. Biotechnol J. 2016 Aug;11(8):1110-7. doi: 10.1002/biot.201600147. Epub 2016 Jun 23. Expresses Cas9 protein, under the control of the TEF1 promoter, and CYC terminator. KanMX resistance pRS414
pRS416gT-GalL-Cas9Cas9 (Synthetic), guide RNA (gRNA) (Synthetic), Tetracycline Repressor (Other)GalL, RPR1 promoter with TetO site, GPM1 promoterURA3 Davis Distinct patterns of Cas9 mismatch tolerance in vitro and in vivo. Nucleic Acids Res. 2016 May 19. pii: gkw417. pRS416 (URA) + RPR1(TetO) promoter with gRNA locus + Tetracycline Repressor + GalL promoter driven Cas9 for yeast expression pRS416
pRCC-KCas9 (Other), empty gRNA cassette (Synthetic)ROX3, SNP52pNeomycin (select with G418) Boles Simplified CRISPR-Cas genome editing for Saccharomyces cerevisiae. J Microbiol Methods. 2016 Aug;127:203-5. doi: 10.1016/j.mimet.2016.06.020. Epub 2016 Jun 17. Expression of Cas9 and gRNA cassette in S. cerevisiae; Kan Resistance pRS42K
  • Tag / Fusion Protein
    • NTS (C terminal on backbone)
  • pRCC-NCas9 (Other), empty gRNA cassette (Synthetic)ROX3, SNP52pNourseothricin Boles Simplified CRISPR-Cas genome editing for Saccharomyces cerevisiae. J Microbiol Methods. 2016 Aug;127:203-5. doi: 10.1016/j.mimet.2016.06.020. Epub 2016 Jun 17. Expression of Cas9 and gRNA cassette in S. cerevisiae; Nourseothricin Resistance pRS42N
  • Tag / Fusion Protein
    • NTS (C terminal on backbone)
  • pDuRCC-KCas9 (Other), empty gRNA cassette (Synthetic), empty gRNA cassette (Synthetic)ROX3, SNP52p, SNP52pNeomycin (select with G418) Boles Simplified CRISPR-Cas genome editing for Saccharomyces cerevisiae. J Microbiol Methods. 2016 Aug;127:203-5. doi: 10.1016/j.mimet.2016.06.020. Epub 2016 Jun 17. Expression of Cas9 and two gRNA cassettes in S. cerevisiae; Kan Resistance pRS42K
  • Tag / Fusion Protein
    • NTS (C terminal on backbone)
  • pDuRCC-NCas9 (Other), empty gRNA cassette (Synthetic), empty gRNA cassette (Synthetic)ROX3, SNP52p, SNP52pNourseothricin Boles Simplified CRISPR-Cas genome editing for Saccharomyces cerevisiae. J Microbiol Methods. 2016 Aug;127:203-5. doi: 10.1016/j.mimet.2016.06.020. Epub 2016 Jun 17. Expression of Cas9 and two gRNA cassettes in S. cerevisiae; Nourseothricin Resistance pRS42N
  • Tag / Fusion Protein
    • NTS (C terminal on backbone)
  • pML104-HygMx4HphMX4 (Other)Hygromycin Wittkopp Patricia Wittkopp yeast CRISPR plasmids (unpublished) Expresses Cas9 and contains guide RNA expression cassette with BclI-SwaI cloning sites for guide sequence cloning; Contains HphMx4 marker for yeast transformation. pRSII426
    pML104-KanMx4KanMX4 (Other)Neomycin (select with G418) Wittkopp Patricia Wittkopp yeast CRISPR plasmids (unpublished) Expresses Cas9 and contains guide RNA expression cassette with BclI-SwaI cloning sites for guide sequence cloning; Contains KanMx4 marker for yeast transformation. pRSII426
    pML104-NatMx3NatMx3 (Other)nourseothricin (nat) Wittkopp Patricia Wittkopp yeast CRISPR plasmids (unpublished) Expresses Cas9 and contains guide RNA expression cassette with BclI-SwaI cloning sites for guide sequence cloning; Contains NatMX3 marker for yeast transformation. pRSII426
    pCfB2312 (TEF1p-Cas9-CYC1t_kanMX)Cas9 (Other)G418 Borodina CRISPR–Cas system enables fast and simple genome editing of industrial Saccharomyces cerevisiae strains Metab Eng Commun 2015 Cas9-carrying vector with kanMX marker p414-TEF1p-Cas9-CYC1t
    pCRISPRyl_AXPCas9UAS1B8-TEF(136)LEU2 Wheeldon Standardized markerless gene integration for pathway engineering in Yarrowia lipolytica. ACS Synth Biol. 2016 Dec 19. Introduces DSB at AXP locus in Y. lipolytica pUC19
    pCRISPRyl_XPR2Cas9UAS1B8-TEF(136)LEU2 Wheeldon Standardized markerless gene integration for pathway engineering in Yarrowia lipolytica. ACS Synth Biol. 2016 Dec 19. Introduces DSB at XPR2 locus in Y. lipolytica pUC19
    pCRISPRyl_A08Cas9UAS1B8-TEF(136)LEU2 Wheeldon Standardized markerless gene integration for pathway engineering in Yarrowia lipolytica. ACS Synth Biol. 2016 Dec 19. Introduces DSB at A08 locus in Y. lipolytica pUC19
    pCRISPRyl_D17Cas9UAS1B8-TEF(136)LEU2 Wheeldon Standardized markerless gene integration for pathway engineering in Yarrowia lipolytica. ACS Synth Biol. 2016 Dec 19. Introduces DSB at D17 locus in Y. lipolytica pUC19
    pCRISPRyl_MFE1Cas9UAS1B8-TEF(136)LEU2 Wheeldon Standardized markerless gene integration for pathway engineering in Yarrowia lipolytica. ACS Synth Biol. 2016 Dec 19. Introduces DSB at MFE1 locus in Y. lipolytica pUC19
    pJB172gRNA targeting pil1 (Schizosaccharomyces pombe)Fluoride Berro Use of a fluoride channel as a new selection marker for fission yeast plasmids and application to fast genome editing with CRISPR/Cas9. Yeast. 2016 Oct;33(10):549-557. doi: 10.1002/yea.3178. Epub 2016 Sep 7. CRISPR/Cas9 in fission yeast using fluoride selection and targetting pil1 pJB166
    pJK-caCas9-NatMX-Neut5LNourseothricin Church caCas9 plasmids (unpublished) caCas9 integrating vector into the Neut5L locus pJK1027
    pJK-caCas9-NatMX-Neut5L-Ade2 driveAde2 gene drive (Other)Nourseothricin Church caCas9 plasmids (unpublished) caCas9 integrating vector into the Neut5L locus with gene drive for targeting Ade2 locus pJK1027
    pJK-caCas9-NatMX-Neut5L-Leu2 driveLeu2 gene drive (Other) Church caCas9 plasmids (unpublished) caCas9 integrating vector into the Neut5L locus with gene drive for targeting Leu2 locus pjk1027
    pWS158Cas9 (Saccharomyces cerevisiae)URA3 Ellis Ellis Lab Yeast CRISPR Plasmids (unpublished) Cas9 gap repair vector - URA3 2 micron
    pWS171Cas9 (Saccharomyces cerevisiae)LEU2 Ellis Ellis Lab Yeast CRISPR Plasmids (unpublished) Cas9 gap repair vector - LEU2 2 micron
    pWS173Cas9 (Saccharomyces cerevisiae)Kanamycin (select with G418) Ellis Ellis Lab Yeast CRISPR Plasmids (unpublished) Cas9 gap repair vector - KanR 2 micron
    pWS175Cas9 (Saccharomyces cerevisiae)Hygromycin Ellis Ellis Lab Yeast CRISPR Plasmids (unpublished) Cas9 gap repair vector - HygR 2 micron
    pADH99US_HIS1, FRT, pENO1, C. albicans Cas9, pMAL2, FLP, NAT 1 of 2Nourseothricin (NAT) Hernday An efficient, rapid, and recyclable system for CRISPR-mediated genome editing in Candida albicans mSphere Apr 2017, 2 (2) e00149-17 NAT-marked CAS9 expression construct; part 1 of 2 of C.alb HIS1-FLP CRISPR system. Use with pADH100-series gRNA expression constructs. pUC19
    pADH137C. albicans LEU2 1 of 2, pENO1, Cas9, NAT 1 of 2Nourseothricin (NAT) Hernday An efficient, rapid, and recyclable system for CRISPR-mediated genome editing in Candida albicans mSphere Apr 2017, 2 (2) e00149-17 NAT-marked CAS9 expression construct; part 1 of 2 of C.alb LEUpOUT CRISPR system. Use with pADH118-series gRNA expression constructs. pUC19
    pADH140C. maltosa LEU2 1 of 2, pENO1, Cas9, NAT 1 of 2Nourseothricin (NAT) Hernday An efficient, rapid, and recyclable system for CRISPR-mediated genome editing in Candida albicans mSphere Apr 2017, 2 (2) e00149-17 NAT-marked CAS9 expression construct; part 1 of 2 of C.mal LEUpOUT CRISPR system. Use with pADH143-series gRNA expression constructs. pUC19
    bRA89Hygromycin Haber Rad51-mediated double-strand break repair and mismatch correction of divergent substrates. Nature. 2017 Apr 20;544(7650):377-380. doi: 10.1038/nature22046. Epub 2017 Apr 12. Cas9 driven by PGK1 promoter. gRNA can be cloned into the BplI sites. HPH marker pRS316
    bRA90LEU2 Haber Rad51-mediated double-strand break repair and mismatch correction of divergent substrates. Nature. 2017 Apr 20;544(7650):377-380. doi: 10.1038/nature22046. Epub 2017 Apr 12. Cas9 driven by PGK1 promoter. gRNA can be cloned into the BplI sites. LEU2 marker pRS316
    bRA66Hygromycin Haber Rad51-mediated double-strand break repair and mismatch correction of divergent substrates. Nature. 2017 Apr 20;544(7650):377-380. doi: 10.1038/nature22046. Epub 2017 Apr 12. GAL1 driven Cas9. gRNA can be cloned into BplI sites. pRS316
    bRA77LEU2 Haber Rad51-mediated double-strand break repair and mismatch correction of divergent substrates. Nature. 2017 Apr 20;544(7650):377-380. doi: 10.1038/nature22046. Epub 2017 Apr 12. GAL1 driven Cas9. gRNA can be cloned into BplI sites. pRS316
    pJH2970HIS3 Haber Cas9-mediated gene editing in Saccharomyces cerevisiae Protocol Exchange (2017) Cas9 driven by PGK1 promoter. gRNA can be cloned into the BplI sites. HIS3MX marker pRS316
    pJH2971Neomycin (select with G418) Haber Cas9-mediated gene editing in Saccharomyces cerevisiae Protocol Exchange (2017) Cas9 driven by PGK1 promoter. gRNA can be cloned into the BplI sites. KANMX marker pRS316
    pJH2972URA3 Haber Cas9-mediated gene editing in Saccharomyces cerevisiae Protocol Exchange (2017) Cas9 driven by PGK1 promoter. gRNA can be cloned into the BplI sites. URA3MX marker pRS316

    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.

    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

    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 Selectable Marker PI Publication Hidden Extra Search Info
    pTDH3-dCas9dCas9TDH3LEU2 Weissman CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes. Cell. 2013 Jul 9. pii: S0092-8674(13)00826-X. doi: 10.1016/j.cell.2013.06.044. Yeast CEN/ARS vector (Leu2) that contains dCas9 fused to NLS controlled by TDH3 promoter pJED103
    pTDH3-dCas9-Mxi1dCas9-Mxi1TDH3LEU2 Weissman CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes. Cell. 2013 Jul 9. pii: S0092-8674(13)00826-X. doi: 10.1016/j.cell.2013.06.044. Yeast CEN/ARS vector (Leu2) that contains dCas9 fused to NLS and Mxi1 domain controlled by TDH3 promoter pJED103
    pJZC518dCas9 (Other)pTdh3LEU2 Qi Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds. Cell. 2014 Dec 18. pii: S0092-8674(14)01570-0. doi: 10.1016/j.cell.2014.11.052. Yeast dCas9 expression plasmid pNH605
    pJZC572sgRNA + 1x com RNA binding moduleSNR52URA3 Qi Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds. Cell. 2014 Dec 18. pii: S0092-8674(14)01570-0. doi: 10.1016/j.cell.2014.11.052. sgRNA with 1x com for yeast cells pRS416
    pTPGI_dCas9Yeast-optimized dCas9 (Saccharomyces cerevisiae)pTPGITRP1 Lu Tunable and Multifunctional Eukaryotic Transcription Factors Based on CRISPR/Cas. ACS Synth Biol. 2013 Sep 11. encodes yeast-optimized dCas9 synthetic transcription factor pRS304
    pRS416-dCas9-Mxi1 + TetR + pRPR1(TetO)-NotI-gRNAdCas9-Mxi1 (Synthetic), Tet Repressor (Other), Structural gRNA for S pyogenes (Synthetic)pTef1, pGPM1, pRPR1(TetO)URA3 Davis Quantitative CRISPR interference screens in yeast identify chemical-genetic interactions and new rules for guide RNA design. Genome Biol. 2016 Mar 8;17(1):45. doi: 10.1186/s13059-016-0900-9. pRS416 Ura marked Cen/Ars plasmid with dCas9-Mxi1 under Tef1 promoter, and tet-incucibile RPR1 promoter with NotI cloning site adjacent to gRNA pRS416
    pCRISPRi_Mxi1_ylCodon optimized dCas9-Mxi1 (Synthetic), sgRNA expression cassette (Synthetic)UAS1B8-TEF(136), SCR1'-tRNALEU2 Wheeldon CRISPRi repression of nonhomologous end-joining for enhanced genome engineering via homologous recombination in Yarrowia lipolytica. Biotechnol Bioeng. 2017 Aug 19. doi: 10.1002/bit.26404. CRISPR-dCas9-Mxi1 vector for Yarrowia lipolytica, expressing dCas9-Mxi1 and AvrII site for sgRNA insertion pUC19
    pCRISPRi_Mxi1_yl_NHEJCodon optimized dCas9-Mxi1 (Synthetic), KU70 sgRNA expression cassette (Synthetic), KU80a sgRNA expression cassette (Synthetic), KU80b sgRNA expression cassette (Synthetic)UAS1B8-TEF(136), SCR1'-tRNA, SCR1'-tRNA, SCR1'-tRNALEU2 Wheeldon CRISPRi repression of nonhomologous end-joining for enhanced genome engineering via homologous recombination in Yarrowia lipolytica. Biotechnol Bioeng. 2017 Aug 19. doi: 10.1002/bit.26404. CRISPRi vector for Yarrowia lipolytica, repressing KU70 and KU80 for enhanced HR pUC19

    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
    pTPGI_dCas9_VP64dCas9_VP64 (codon-optimized for expression in S. cerevisiae) (Saccharomyces cerevisiae)pTPGI (galactose+aTc inducible)TRP1 Lu Tunable and Multifunctional Eukaryotic Transcription Factors Based on CRISPR/Cas. ACS Synth Biol. 2013 Sep 11. encodes yeast-optimized dCas9_VP64 synthetic transcription factor pTPGI (pRS304)
    pJZC519dCas9-VP64 (Other)pTdh3LEU2 Qi Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds. Cell. 2014 Dec 18. pii: S0092-8674(14)01570-0. doi: 10.1016/j.cell.2014.11.052. Yeast dCas9-VP64 expression plasmid pNH605
    pJZC620MCP-VP64, PCP-VP64, dCas9pAdh, pAdh, pTdh3LEU2 Qi Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds. Cell. 2014 Dec 18. pii: S0092-8674(14)01570-0. doi: 10.1016/j.cell.2014.11.052. Expresses dCas9, MCP-VP64, and PCP-VP64 in Yeast cells pNH605
    pJZC638MCP-VP64, dCas9 (Other)pAdh, pGal10LEU2 Qi Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds. Cell. 2014 Dec 18. pii: S0092-8674(14)01570-0. doi: 10.1016/j.cell.2014.11.052. Expresses MCP-VP64 and dCas9 in Yeast cells pNH605
    pJZC545sgRNA + 1x MS2 binding moduleSNR52URA3 Qi Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds. Cell. 2014 Dec 18. pii: S0092-8674(14)01570-0. doi: 10.1016/j.cell.2014.11.052. sgRNA with 1x MS2 for yeast cells pRS416
    pJZC583sgRNA + 2x MS2 binding moduleSNR52URA3 Qi Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds. Cell. 2014 Dec 18. pii: S0092-8674(14)01570-0. doi: 10.1016/j.cell.2014.11.052. sgRNA with 2x MS2 for yeast cells pRS416
    pJZC548sgRNA + 1x PP7SNR52URA3 Qi Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds. Cell. 2014 Dec 18. pii: S0092-8674(14)01570-0. doi: 10.1016/j.cell.2014.11.052. sgRNA with 1x PP7 for yeast cells pRS416
    pJZC603sgRNA + 2x PP7 RNA binding moduleSNR52URA3 Qi Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds. Cell. 2014 Dec 18. pii: S0092-8674(14)01570-0. doi: 10.1016/j.cell.2014.11.052. sgRNA with 2x PP7 for yeast cells pRS416
    pJZC572sgRNA + 1x com RNA binding moduleSNR52URA3 Qi Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds. Cell. 2014 Dec 18. pii: S0092-8674(14)01570-0. doi: 10.1016/j.cell.2014.11.052. sgRNA with 1x com for yeast cells pRS416
    pJZC593sgRNA + MS2-PP7 RNA binding moduleSNR52URA3 Qi Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds. Cell. 2014 Dec 18. pii: S0092-8674(14)01570-0. doi: 10.1016/j.cell.2014.11.052. sgRNA with MS2-PP7 for yeast cells pRS416
    pJZC625sgRNA +1x MS2, Pol II promoter with ribozyme cleavagepAdhURA3 Qi Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds. Cell. 2014 Dec 18. pii: S0092-8674(14)01570-0. doi: 10.1016/j.cell.2014.11.052. sgRNA with 1x MS2, Pol II promoter with ribozyme-gRNA-ribozyme design for yeast cells pSV616
    pJZC595MCP-VP64, dCas9pAdh, Tdh3LEU2 Qi Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds. Cell. 2014 Dec 18. pii: S0092-8674(14)01570-0. doi: 10.1016/j.cell.2014.11.052. Expresses MCP-VP64 and dCas9 in Yeast cells pNH605
    pAG414GPD-dCas9-VPRdCas9-VPR (Saccharomyces cerevisiae)GPDTRP1 Church Highly efficient Cas9-mediated transcriptional programming. Nat Methods. 2015 Mar 2. doi: 10.1038/nmeth.3312. pAG414 series plasmid with GPD promoter driving expression of dCas9-VPR; cerevisiae vector pAG414GPD
    pTPGI_dCas9Yeast-optimized dCas9 (Saccharomyces cerevisiae)pTPGITRP1 Lu Tunable and Multifunctional Eukaryotic Transcription Factors Based on CRISPR/Cas. ACS Synth Biol. 2013 Sep 11. encodes yeast-optimized dCas9 synthetic transcription factor pRS304
    pRS416-Gal4-dCas9-VP64Gal4-dCas9-VP64 (Saccharomyces cerevisiae)Tef1URA3 Davis Dissecting the Genetic Basis of a Complex cis-Regulatory Adaptation. PLoS Genet. 2015 Dec 29;11(12):e1005751. doi: 10.1371/journal.pgen.1005751. eCollection 2015 Dec. This plasmid contains a Cas9 Activator for yeast. The activator is about 1.2-1.8 X more potent than just dCas9-VP64 fusion in yeast. It is on a single copy CEN/ARS plasmid with Ura marker, pRS416. pRS416

    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 Selectable Marker PI Publication Hidden Extra Search Info
    3xFLAG-dCas9/pTEF1p-CYC1t3xFLAG-dCas9 (Synthetic)TEF1 promoterTRP1 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. Expresses 3xFLAG-dCas9 in budding yeast for enChIP analysis to purify specific genomic regions of interest. p414

    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)
    pRPR1_gRNA_
    handle_RPR1t
    pRPR1 HindIII S. cerevisiae LEU2 none, need Cas9 plasmid Lu

    Last Updated: February 20, 2015

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