CRISPR/Cas9 plasmids for use in Yeast

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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 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-CYC1t	Human Optimized S. pyogenes Cas9 (Homo sapiens)	TEF1 promoter	TRP1	Church	Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res	p414
	p415-GalL-Cas9-CYC1t	Human Optimized S. pyogenes Cas9 (Homo sapiens)	GalL	LEU2	Church	Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res	p415
	pACT2-CAS9c	Cas9c (Synthetic)	yeast ADH1 promoter	LEU2	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
	pMZ222	Cas9 (Other)	adh1	LEU2	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
	pMZ374	Cas9 (Other), rrk1:sgRNA (Other)	adh1, rrk1	ura4	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
	pCT	iCas9 (Other), tracrRNA (Other)	TEF1p, RPR1p	LEU2	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
	pCRCT	iCas9 (Other), tracrRNA (Other)	TEF1p, RPR1p	URA3	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-NAT	Human-optimized S. pyogenes Cas9 with Clonnat marker gene expression cassette		Nourseothricin(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
	pML104	Cas9 (Saccharomyces cerevisiae), single guide RNA expression cassette (Saccharomyces cerevisiae)	pTDH3, pSNR52	URA3	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
	pML107	Cas9 (Saccharomyces cerevisiae), single guide RNA expression cassette (Saccharomyces cerevisiae)	pTDH3, pSNR52	LEU2	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
	pJH001	Cas9 (Saccharomyces cerevisiae), Empty		LEU2	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-CYC1t	humanized 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
	pCRISPRyl	Codon optimized Cas9 from S. pyogenes (Synthetic), sgRNA expression cassette (Synthetic)	UAS1B8-TEF(136), SCR1'-tRNA	LEU2	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
	pMZ376	Cas9 (Other), rrk1:sgRNA (Other)	adh1, rrk1	Neomycin (select with G418)	Zaratiegui	sgRNA/Cas9 (unpublished)	Combined sgRNA/Cas9 vector with KanMX marker pMZ374
	pMZ377	Cas9 (Other), rrk1:sgRNA (Other)	adh1, rrk1	LEU2	Zaratiegui	sgRNA/Cas9 (unpublished)	Combined sgRNA/Cas9 vector with LEU2 marker pMZ374
	pMZ379	Cas9 (Other), rrk1:sgRNA (Other)	adh1, rrk1	nourseothricin	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-Cas9	Cas9 (Synthetic), guide RNA (gRNA) (Synthetic), Tetracycline Repressor (Other)	GalL, RPR1 promoter with TetO site, GPM1 promoter	URA3	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-K	Cas9 (Other), empty gRNA cassette (Synthetic)	ROX3, SNP52p	Neomycin (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-N	Cas9 (Other), empty gRNA cassette (Synthetic)	ROX3, SNP52p	Nourseothricin	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-K	Cas9 (Other), empty gRNA cassette (Synthetic), empty gRNA cassette (Synthetic)	ROX3, SNP52p, SNP52p	Neomycin (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-N	Cas9 (Other), empty gRNA cassette (Synthetic), empty gRNA cassette (Synthetic)	ROX3, SNP52p, SNP52p	Nourseothricin	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-HygMx4	HphMX4 (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-KanMx4	KanMX4 (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-NatMx3	NatMx3 (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_AXP	Cas9	UAS1B8-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_XPR2	Cas9	UAS1B8-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_A08	Cas9	UAS1B8-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_D17	Cas9	UAS1B8-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_MFE1	Cas9	UAS1B8-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
	pJB172	gRNA 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
	pADH99	US_HIS1, FRT, pENO1, C. albicans Cas9, pMAL2, FLP, NAT 1 of 2		Nourseothricin (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
	pADH137	C. albicans LEU2 1 of 2, pENO1, Cas9, NAT 1 of 2		Nourseothricin (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
	pADH140	C. maltosa LEU2 1 of 2, pENO1, Cas9, NAT 1 of 2		Nourseothricin (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
	bRA89			Hygromycin	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
	bRA90			LEU2	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
	bRA66			Hygromycin	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
	bRA77			LEU2	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
	pJH2970			HIS3	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
	pJH2971			Neomycin (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
	pJH2972			URA3	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
	79616	pRS415_pGal-nCas9(H840A)	Expresses nCas9(H840A) in yeast cells	SpCas9 (Other)	pGal1	LEU2	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
	79617	pRS315e_pGal-nCas9(D10A)-PmCDA1	Expresses nCas9(D10A)-PmCDA1 in yeast cells	SpCas9 (Other)	pGal1	LEU2	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
	79618	pRS415_pGal-nCas9(D10A)	Expresses nCas9(D10A) in yeast cells	SpCas9 (Other)	pGal1	LEU2	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-dCas9	dCas9	TDH3	LEU2	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-Mxi1	dCas9-Mxi1	TDH3	LEU2	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
	pJZC518	dCas9 (Other)	pTdh3	LEU2	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
	pJZC572	sgRNA + 1x com RNA binding module	SNR52	URA3	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_dCas9	Yeast-optimized dCas9 (Saccharomyces cerevisiae)	pTPGI	TRP1	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-gRNA	dCas9-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_yl	Codon optimized dCas9-Mxi1 (Synthetic), sgRNA expression cassette (Synthetic)	UAS1B8-TEF(136), SCR1'-tRNA	LEU2	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_NHEJ	Codon 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'-tRNA	LEU2	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_VP64	dCas9_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)
	pJZC519	dCas9-VP64 (Other)	pTdh3	LEU2	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
	pJZC620	MCP-VP64, PCP-VP64, dCas9	pAdh, pAdh, pTdh3	LEU2	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
	pJZC638	MCP-VP64, dCas9 (Other)	pAdh, pGal10	LEU2	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
	pJZC545	sgRNA + 1x MS2 binding module	SNR52	URA3	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
	pJZC583	sgRNA + 2x MS2 binding module	SNR52	URA3	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
	pJZC548	sgRNA + 1x PP7	SNR52	URA3	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
	pJZC603	sgRNA + 2x PP7 RNA binding module	SNR52	URA3	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
	pJZC572	sgRNA + 1x com RNA binding module	SNR52	URA3	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
	pJZC593	sgRNA + MS2-PP7 RNA binding module	SNR52	URA3	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
	pJZC625	sgRNA +1x MS2, Pol II promoter with ribozyme cleavage	pAdh	URA3	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
	pJZC595	MCP-VP64, dCas9	pAdh, Tdh3	LEU2	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-VPR	dCas9-VPR (Saccharomyces cerevisiae)	GPD	TRP1	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_dCas9	Yeast-optimized dCas9 (Saccharomyces cerevisiae)	pTPGI	TRP1	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-VP64	Gal4-dCas9-VP64 (Saccharomyces cerevisiae)	Tef1	URA3	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-CYC1t	3xFLAG-dCas9 (Synthetic)	TEF1 promoter	TRP1	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