CRISPR Plasmids: Yeast

The following CRISPR plasmids have been designed for use in yeast.

Cut

Fully functional CRISPR/Cas enzymes will introduce a double-strand break (DSB) at a specific location based on a 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. Indels often lead to frameshifts, creating loss of function alleles.

To introduce specific genomic changes, researchers use ssDNA or dsDNA repair templates with homology to the DNA flanking the DSB and a specific edit close to the gRNA PAM site. When a repair template is present, the cell may repair a DSB using homology-directed repair (HDR) instead of NHEJ. In most experimental systems, HDR occurs at a much lower efficiency than NHEJ.

	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 (aka GAP promoter), 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
	pJK-caCas9-NatMX-Neut5L			Nourseothricin	Church	A CRISPR-Cas9-based gene drive platform for genetic interaction analysis in Candida albicans. Nat Microbiol. 2018 Jan;3(1):73-82. doi: 10.1038/s41564-017-0043-0. Epub 2017 Oct 23.	caCas9 integrating vector into the Neut5L locus pJK1027
	pJK-caCas9-NatMX-Neut5L-Ade2 drive	Ade2 gene drive (Other)		Nourseothricin	Church	A CRISPR-Cas9-based gene drive platform for genetic interaction analysis in Candida albicans. Nat Microbiol. 2018 Jan;3(1):73-82. doi: 10.1038/s41564-017-0043-0. Epub 2017 Oct 23.	caCas9 integrating vector into the Neut5L locus with gene drive for targeting Ade2 locus pJK1027
	pJK-caCas9-NatMX-Neut5L-Leu2 drive	Leu2 gene drive (Other)			Church	A CRISPR-Cas9-based gene drive platform for genetic interaction analysis in Candida albicans. Nat Microbiol. 2018 Jan;3(1):73-82. doi: 10.1038/s41564-017-0043-0. Epub 2017 Oct 23.	caCas9 integrating vector into the Neut5L locus with gene drive for targeting Leu2 locus pjk1027
	pWS173	Cas9 (Saccharomyces cerevisiae)		Kanamycin (select with G418)	Ellis	Ellis Lab Yeast CRISPR Plasmids (unpublished)	Cas9 gap repair vector - KanR 2 micron
	pWS174	Cas9 (Saccharomyces cerevisiae)		Nourseothricin	Ellis	Ellis Lab Yeast CRISPR Plasmids (unpublished)	Cas9 gap repair vector - NatR 2 micron
	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
	pDHt/sk-PC	Cas9 (Other)	Ppdc	Hygromycin	Zhou	Efficient genome editing in filamentous fungus Trichoderma reesei using the CRISPR/Cas9 system. Cell Discov. 2015 May 12;1:15007. doi: 10.1038/celldisc.2015.7. eCollection 2015.	Expression of codon-optimized Cas9 with Ppdc promoter in Trichoderma reesei pDHt/sk
	pDHt/sk-PE	Cas9-eGFP (Other)	Ppdc	Hygromycin	Zhou	Efficient genome editing in filamentous fungus Trichoderma reesei using the CRISPR/Cas9 system. Cell Discov. 2015 May 12;1:15007. doi: 10.1038/celldisc.2015.7. eCollection 2015.	Expression of codon-optimized Cas9-eGFP fusion protein with Ppdc promoter in Trichoderma reesei pDHt/sk Tag / Fusion Protein EGFP (C terminal on insert)
	pDHt/sk-CC	Cas9 (Other)	Pcbh1	Hygromycin	Zhou	Efficient genome editing in filamentous fungus Trichoderma reesei using the CRISPR/Cas9 system. Cell Discov. 2015 May 12;1:15007. doi: 10.1038/celldisc.2015.7. eCollection 2015.	Expression of codon-optimized Cas9 with Pcbh1 promoter in Trichoderma reesei pDHt/sk
	pDHt/sk-CE	Cas9-eGFP (Other)	Pcbh2	Hygromycin	Zhou	Efficient genome editing in filamentous fungus Trichoderma reesei using the CRISPR/Cas9 system. Cell Discov. 2015 May 12;1:15007. doi: 10.1038/celldisc.2015.7. eCollection 2015.	Expression of codon-optimized Cas9-eGFP fusion protein with Pcbh1 promoter in Trichoderma reesei pDHt/sk Tag / Fusion Protein EGFP (C terminal on insert)
	pYZ033			Spura4	Boeke	Construction of Designer Selectable Marker Deletions with CRISPR-Cas9 Toolbox in Schizosaccharomyces pombe and Optimized Design of Common Entry Vectors. G3 (Bethesda). 2018 Jan 10. pii: g3.117.300363. doi: 10.1534/g3.117.300363.	Entry vector for S. pombe CRISPR-Cas9 system. The gRNA can be integrated easily through Gibson Assembly with the plasmid backbone digested by Not1 pMZ374
	pYZ146	gRNA targeting Sp.leu1 (Schizosaccharomyces pombe)		Spura4	Boeke	Construction of Designer Selectable Marker Deletions with CRISPR-Cas9 Toolbox in Schizosaccharomyces pombe and Optimized Design of Common Entry Vectors. G3 (Bethesda). 2018 Jan 10. pii: g3.117.300363. doi: 10.1534/g3.117.300363.	Cas9-gRNA leu1 plasmid for leu1 deletion in S. pombe pYZ033 leu1 SPBC1A4.02c
	pYZ164	gRNA targeting Sp.his3 (Schizosaccharomyces pombe)		Spura4	Boeke	Construction of Designer Selectable Marker Deletions with CRISPR-Cas9 Toolbox in Schizosaccharomyces pombe and Optimized Design of Common Entry Vectors. G3 (Bethesda). 2018 Jan 10. pii: g3.117.300363. doi: 10.1534/g3.117.300363.	Cas9-gRNA his3 plasmid for his3 deletion in S. pombe pYZ033 his3 SPBC11B10.02c
	pYZ173	gRNA targeting Sp.lys9 (Schizosaccharomyces pombe)		Spura4	Boeke	Construction of Designer Selectable Marker Deletions with CRISPR-Cas9 Toolbox in Schizosaccharomyces pombe and Optimized Design of Common Entry Vectors. G3 (Bethesda). 2018 Jan 10. pii: g3.117.300363. doi: 10.1534/g3.117.300363.	Cas9-gRNA lys9 plasmid for lys9 deletion in S. pombe pYZ033 SPBC3B8.03 SPBC3B8.03
	pDB4279	Cas9 (Other)			Du	A Cloning-Free Method for CRISPR/Cas9-Mediated Genome Editing in Fission Yeast. G3 (Bethesda). 2018 Apr 27. pii: g3.118.200164. doi: 10.1534/g3.118.200164.	A control plasmid containing the intact bsdMX marker pMZ374 (Addgene 59896)
	pDB4280	Cas9 (Other)			Du	A Cloning-Free Method for CRISPR/Cas9-Mediated Genome Editing in Fission Yeast. G3 (Bethesda). 2018 Apr 27. pii: g3.118.200164. doi: 10.1534/g3.118.200164.	A Cas9-encoding plasmid containing the 5' portion of the ura4 marker and the rrk1 promoter/leader. When linearized by NotI, it serves as the gapped vector in the split-ura4 system pMZ374 (Addgene 59896)
	pDB4281	Cas9 (Other)			Du	A Cloning-Free Method for CRISPR/Cas9-Mediated Genome Editing in Fission Yeast. G3 (Bethesda). 2018 Apr 27. pii: g3.118.200164. doi: 10.1534/g3.118.200164.	A Cas9-encoding plasmid containing the 5' portion of the bsdMX marker and the rrk1 promoter/leader. When linearized by NotI, it serves as the gapped vector in the split-bsdMX system pMZ374 (Addgene 59896)
	pIW447-CRISPR XYL2	Codon optimized Cas9 (Synthetic), sgRNA expression cassette (Synthetic)		URA3	Wheeldon	CRISPR-Cas9-enabled genetic disruptions for understanding ethanol and ethyl acetate biosynthesis in Kluyveromyces marxianus. Biotechnol Biofuels. 2017 Jun 24;10:164. doi: 10.1186/s13068-017-0854-5. eCollection 2017.	K. marxianus CRISPR Plasmid for XYL2 editing pJSK316-GPD
	pIW601-KmCRISPR	Codon optimized Cas9 (Synthetic), sgRNA expression cassette (Synthetic)		URA3	Wheeldon	CRISPR-Cas9-enabled genetic disruptions for understanding ethanol and ethyl acetate biosynthesis in Kluyveromyces marxianus. Biotechnol Biofuels. 2017 Jun 24;10:164. doi: 10.1186/s13068-017-0854-5. eCollection 2017.	K. marxianus CRISPR Plasmid for sgRNA cloning pJSK316-GPD
	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
	pUDP004		ScTDH3	amdS (acetamidase gene conferring growth on acetamide)	Daran	CRISPR-Cas9 mediated gene deletions in lager yeast Saccharomyces pastorianus. Microb Cell Fact. 2017 Dec 5;16(1):222. doi: 10.1186/s12934-017-0835-1.	E. coli/S. cerevisiae shuttle vector carrying amd S marker and Spcas9D147Y P411T allowing cloning of ribozyme flanked g-RNA for Cas9 editing (HH-gRNA-HDV) not applicable
	pUDP012	HH-gRNA-HDV targetting SeILV6 in S. pastorianus (Saccharomyces cerevisiae)	ScTDH3, ScTDH3	amdS (acetamidase gene conferring growth on acetamide)	Daran	CRISPR-Cas9 mediated gene deletions in lager yeast Saccharomyces pastorianus. Microb Cell Fact. 2017 Dec 5;16(1):222. doi: 10.1186/s12934-017-0835-1.	E. coli/S. cerevisiae shuttle vector carrying amdS andSpcas9D147Y P411T and expressing a ribozyme flanked g-RNA for Cas9 editing targeting the gene SeILV6 and in S. pastorianus (HH-gRNASeILV6-HDV) pUDP004
	pUDP044	polycistronic HH-gRNA-HDV-HH-gRNA-HDV array targetting SeATF1 and 2 in S. pastorianus (Saccharomyces cerevisiae)	ScTDH3, ScTDH3	amdS (acetamidase gene conferring growth on acetamide)	Daran	CRISPR-Cas9 mediated gene deletions in lager yeast Saccharomyces pastorianus. Microb Cell Fact. 2017 Dec 5;16(1):222. doi: 10.1186/s12934-017-0835-1.	pUDP004 expressing a polycistronic g-RNA array for Cas9 editing targeting the genes SeATF1 and SeATF2 and Spcas9D147Y P411T in S. pastorianus (HH-gRNASeATF1-HDV-linker-HH-gRNASeATF2-HDV) pUDP004
	pCSN061	S. pyogenes Cas9 codon optimized for expression in S. cerevisiae., KanMX marker expression cassette. (Other)	Heterologous promoter from K. lactis (KLLA0F20031g), Heterologous TEF1 promoter from A. gossypii.	TRP1 ; KanMX	Verwaal	CRISPR/Cpf1 enables fast and simple genome editing of Saccharomyces cerevisiae. Yeast. 2017 Sep 8. doi: 10.1002/yea.3278.	Single copy yeast plasmid expressing Cas9 from Streptococcus pyogenes (SpCas9), codon optimized for expression in Saccharomyces cerevisiae. pRS414 NEWENTRY
	pCSN067	Cpf1 from Lachnospiraceae bacterium ND2006 (LbCpf1) codon optimized for expression in S. cerevisiae. (Other), KanMX marker expression cassette. (Other)	Heterologous promoter from K. lactis (KLLA0F20031g)., Heterologous TEF1 promoter from A. gossypii.	TRP1 ; KanMX	Verwaal	CRISPR/Cpf1 enables fast and simple genome editing of Saccharomyces cerevisiae. Yeast. 2017 Sep 8. doi: 10.1002/yea.3278.	Single copy yeast plasmid expressing Cpf1 from Lachnospiraceae bacterium ND2006 (LbCpf1), codon optimized for expression in Saccharomyces cerevisiae. pRS414
	pCSN068	Cpf1 from Francisella novicida U112 (FnCpf1) codon optimized for expression in S. cerevisiae. (Other), KanMX marker expression cassette. (Other)	Heterologous promoter from K. lactis (KLLA0F20031g)., Heterologous TEF1 promoter from A. gossypii.	TRP1 ; KanMX	Verwaal	CRISPR/Cpf1 enables fast and simple genome editing of Saccharomyces cerevisiae. Yeast. 2017 Sep 8. doi: 10.1002/yea.3278.	Single copy yeast plasmid expressing Cpf1 from Francisella novicida U112 (FnCpf1), codon optimized for expression in Saccharomyces cerevisiae. pRS414
	pYZ292			LEU2 ; Sc	Boeke	Construction of Designer Selectable Marker Deletions with CRISPR-Cas9 Toolbox in Schizosaccharomyces pombe and Optimized Design of Common Entry Vectors. G3 (Bethesda). 2018 Jan 10. pii: g3.117.300363. doi: 10.1534/g3.117.300363.	S. pombe gRNA entry vector with Cas9 - ScLEU2 marker pYZ292
	pYZ293			Sp leu1	Boeke	Construction of Designer Selectable Marker Deletions with CRISPR-Cas9 Toolbox in Schizosaccharomyces pombe and Optimized Design of Common Entry Vectors. G3 (Bethesda). 2018 Jan 10. pii: g3.117.300363. doi: 10.1534/g3.117.300363.	S. pombe gRNA entry vector with Cas9 - Sp leu1 marker pYZ293
	pYZ294			kanMX	Boeke	Construction of Designer Selectable Marker Deletions with CRISPR-Cas9 Toolbox in Schizosaccharomyces pombe and Optimized Design of Common Entry Vectors. G3 (Bethesda). 2018 Jan 10. pii: g3.117.300363. doi: 10.1534/g3.117.300363.	S. pombe gRNA entry vector with Cas9 - KanMX marker pYZ294
	pYZ300			Sp his3	Boeke	Construction of Designer Selectable Marker Deletions with CRISPR-Cas9 Toolbox in Schizosaccharomyces pombe and Optimized Design of Common Entry Vectors. G3 (Bethesda). 2018 Jan 10. pii: g3.117.300363. doi: 10.1534/g3.117.300363.	S. pombe gRNA entry vector with Cas9 - Sp his3 marker pYZ300
	pYZ301			Sp lys9	Boeke	Construction of Designer Selectable Marker Deletions with CRISPR-Cas9 Toolbox in Schizosaccharomyces pombe and Optimized Design of Common Entry Vectors. G3 (Bethesda). 2018 Jan 10. pii: g3.117.300363. doi: 10.1534/g3.117.300363.	S. pombe gRNA entry vector with Cas9 - Sp lys9 marker pYZ301
	pUDE731	Fncpf1 (Other)	TEF1	URA3	Daran	FnCpf1, a novel and efficient genome editing tool for Saccharomyces cerevisiae. Nucleic Acids Research (2017), gkx1007	S. cerevisiae Episomal plasmid harboring Fncpf1 under the control of TDH3p pMEL10
	pUDC175	Fncpf1 (Other)	TEF1	TRP1	Daran	FnCpf1, a novel and efficient genome editing tool for Saccharomyces cerevisiae. Nucleic Acids Research (2017), gkx1007	S. cerevisae centromic plasmid harboring Fncpf1 under control of TEF1 promoter p414TEF1
	pUDP002	hph (Other), Spcas9 D147Y P411T (Other)	Ashbya gossypii (Eremothecium gossypii) TEF1, Arxula adeninivorans TEF1	Hygromycin	Daran	Genome editing in Kluyveromyces and Ogataea yeasts using a broad-host-range Cas9/gRNA co-expression plasmid. FEMS Yeast Res. 2018 May 1;18(3). pii: 4847887. doi: 10.1093/femsyr/foy012.	Broad-host-range Cas9/gRNA co-expression backbone plasmid (no gRNA) not applicable
	BB3cN_pGAP_23*_pLAT1_Cas9	HH - FS23 linker - HDV (Other), human codon-optimized hcas9 sequence fused to a nuclear localization signal (NLS) (Homo sapiens)		NTC	Gasser	CRISPR/Cas9-mediated homology directed genome editing in Pichia pastoris (unpublished)	hCas9 under control of LAT1 for direct cloning of HH-sgRNA-HDV PCR products for episomal expression in P. pastoris and selection on NTC Episomal ARS/CEN Backbone
	BB3cH_pGAP_23*_pLAT1_Cas9	HH - FS23 linker - HDV (Other), human codon-optimized hcas9 sequence fused to a nuclear localization signal (NLS) (Homo sapiens)		Hygromycin	Gasser	CRISPR/Cas9-mediated homology directed genome editing in Pichia pastoris (unpublished)	hCas9 under control of LAT1 for direct cloning of HH-sgRNA-HDV PCR products for episomal expression in P. pastoris and selection on Hyg Episomal ARS/CEN Backbone
	BB3cK_pGAP_23*_pLAT1_Cas9	HH - FS23 linker - HDV (Other), human codon-optimized hcas9 sequence fused to a nuclear localization signal (NLS) (Homo sapiens)		Neomycin (select with G418)	Gasser	CRISPR/Cas9-mediated homology directed genome editing in Pichia pastoris (unpublished)	hCas9 under control of LAT1 for direct cloning of HH-sgRNA-HDV PCR products for episomal expression in P. pastoris and selection on G418 Episomal ARS/CEN Backbone
	BB3cK_pGAP_23*_pTEF_Cas9	HH - FS23 linker - HDV (Other), human codon-optimized hcas9 sequence fused to a nuclear localization signal (NLS) (Homo sapiens)		Neomycin (select with G418)	Gasser	CRISPR/Cas9-mediated homology directed genome editing in Pichia pastoris (unpublished)	hCas9 under control of Tef1 for direct cloning of HH-sgRNA-HDV PCR products and episomal expression in P. pastoris and G418 selection Episomal ARS/CEN Backbone
	BB3cK_pGAP_23*_pPFK300_Cas9	HH - FS23 linker - HDV (Other), human codon-optimized hcas9 sequence fused to a nuclear localization signal (NLS) (Homo sapiens)		Neomycin (select with G418)	Gasser	CRISPR/Cas9-mediated homology directed genome editing in Pichia pastoris (unpublished)	hCas9 under control of pPFK300 for direct cloning of HH-sgRNA-HDV PCR products for episomal expression in P. pastoris and selection on G418 Episomal ARS/CEN Backbone
	BB3cN_pGAP_23*_pPFK300_Cas9	HH - FS23 linker - HDV (Other), human codon-optimized hcas9 sequence fused to a nuclear localization signal (NLS) (Homo sapiens)		NTC	Gasser	CRISPR/Cas9-mediated homology directed genome editing in Pichia pastoris (unpublished)	hCas9 under control of pPFK300 for direct cloning of HH-sgRNA-HDV PCR products for episomal expression in P. pastoris and selection on NTC Episomal ARS/CEN Backbone
	BB3cH_pGAP_23*_pPFK300_Cas9	HH - FS23 linker - HDV (Other), human codon-optimized hcas9 sequence fused to a nuclear localization signal (NLS) (Homo sapiens)		Hygromycin	Gasser	CRISPR/Cas9-mediated homology directed genome editing in Pichia pastoris (unpublished)	hCas9 under control of pPFK300 for direct cloning of HH-sgRNA-HDV PCR products for episomal expression in P. pastoris and selection on Hyg Episomal ARS/CEN Backbone
	pCfB4906	Cas9 (Other)		Hygromycin	Borodina	EasyCloneYALI: CRISPR/Cas9-Based Synthetic Toolbox for Engineering of the Yeast Yarrowia lipolytica. Biotechnol J. 2018 Jan 29. doi: 10.1002/biot.201700543.	EasyCloneYALI system-based yeast integrative vector expressing Cas9 carrying hygromycin resistance marker, integration into Yarrowia lipolytica chromosomal location IntB, amp resistance Unknown
	pCfB6364	Cas9 (Other)		D-serine marker; enables growth on D-serine	Borodina	EasyCloneYALI: CRISPR/Cas9-Based Synthetic Toolbox for Engineering of the Yeast Yarrowia lipolytica. Biotechnol J. 2018 Jan 29. doi: 10.1002/biot.201700543.	EasyCloneYALI system-based yeast integrative vector expressing Cas9 carrying D-serine marker, integration into Yarrowia lipolytica ku70 locus, amp resistance Unknown
	pHCas9-Nours	Cas9 (Other)	TEF1	URA3	Yang	Unraveling the genetic basis of fast l-arabinose consumption on top of recombinant xylose-fermenting Saccharomyces cerevisiae. Biotechnol Bioeng. 2018 Sep 10. doi: 10.1002/bit.26827.	Escherichia coli- S. cerevisiae shuttle plasmid harbors a Cas9 gene, a natMX6 and URA3 selection markers used in S. cerevisiae pSH47
	pV1025	CaCas9 (Synthetic)		Ampicillin	Fink	A CRISPR system permits genetic engineering of essential genes and gene families. Sci Adv. 2015;1(3):e1500248.	Expression of Candida albicans codon-optimized Cas9 (CaCas9) - integrates at ENO1 (Part of Duet system) pV941
	pV1093	CaCas9/sgRNA-BsmBI stuffer (Synthetic)		clonNAT	Fink	A CRISPR system permits genetic engineering of essential genes and gene families. Sci Adv. 2015;1(3):e1500248.	CaCas9/gRNA Solo entry plasmid for cloning guides - contains stuffer with BsmBI sites - integrates at ENO1 pV1088/pV1090
	pV1200	CaCas9/sgRNA-BsmBI stuffer (Synthetic)		Ampicillin	Fink	A CRISPR system permits genetic engineering of essential genes and gene families. Sci Adv. 2015;1(3):e1500248.	CaCas9/gRNA Solo entry plasmid for cloning guides - contains stuffer with BsmBI sites - integrates at ENO1 - Recyclable for serial mutagenesis (Sap2-FLP) pV1025/pV1093
	pV1393	CaCas9/sgRNA-BsmBI stuffer (Synthetic)		Ampicillin	Fink	New CRISPR Mutagenesis Strategies Reveal Variation in Repair Mechanisms among Fungi. mSphere. 2018 Apr 25;3(2). pii: 3/2/e00154-18. doi: 10.1128/mSphere.00154-18. Print 2018 Apr 25.	CaCas9/gRNA Solo entry plasmid for cloning guides - contains stuffer with BsmBI sites - inserts at Neut5L - Recyclable for serial mutagenesis (Sap2-FLP) pV1390
	pV1539	CaCas9/sgRNA-BsmBI stuffer (Synthetic)		Ampicillin	Fink	New CRISPR Mutagenesis Strategies Reveal Variation in Repair Mechanisms among Fungi. mSphere. 2018 Apr 25;3(2). pii: 3/2/e00154-18. doi: 10.1128/mSphere.00154-18. Print 2018 Apr 25.	Genedrive entry vector for Candida albicans pV1524
	pV1326	CaCas9/sgRNA-BsmBI stuffer (Synthetic)		URA3 ; clonNAT	Fink	New CRISPR Mutagenesis Strategies Reveal Variation in Repair Mechanisms among Fungi. mSphere. 2018 Apr 25;3(2). pii: 3/2/e00154-18. doi: 10.1128/mSphere.00154-18. Print 2018 Apr 25.	S. cerevisiae and C. glabrata Solo CRISPR vector, marked with URA3 and NatR pRS416
	pV1382	CaCas9/sgRNA-BsmBI stuffer (Synthetic)		URA3 ; clonNAT	Fink	New CRISPR Mutagenesis Strategies Reveal Variation in Repair Mechanisms among Fungi. mSphere. 2018 Apr 25;3(2). pii: 3/2/e00154-18. doi: 10.1128/mSphere.00154-18. Print 2018 Apr 25.	S. cerevisiae and C. glabrata Solo CRISPR vector, marked with URA3 and NatR pRS416
	pV1464	CaCas9/sgRNA-BsmBI stuffer (Synthetic)		URA3 ; clonNAT	Fink	New CRISPR Mutagenesis Strategies Reveal Variation in Repair Mechanisms among Fungi. mSphere. 2018 Apr 25;3(2). pii: 3/2/e00154-18. doi: 10.1128/mSphere.00154-18. Print 2018 Apr 25.	N. castellii Solo CRISPR vector, marked with URA3 and NatR pRS416
	pZS157 CRISPEY RT/Cas9	SpCas9, Ec86-RT (Other)	GAL1-GAL10	HIS3	Fraser	Functional Genetic Variants Revealed by Massively Parallel Precise Genome Editing. Cell. 2018 Sep 18. pii: S0092-8674(18)31118-8. doi: 10.1016/j.cell.2018.08.057.	Yeast Integration Plasmid for galactose inducible Ec86-RT and SpCas9 expression pRS403
	eGFP L202 gRNA	eGFP L202 gRNA (Other)	U6		Harris	A panel of eGFP reporters for single base editing by APOBEC-Cas9 editosome complexes. Sci Rep. 2019 Jan 24;9(1):497. doi: 10.1038/s41598-018-36739-9.	gRNA that guides Cas9 and APOBEC complexes to L202 in eGFP. MLM3636
	eGFP L138 gRNA	eGFP L138 gRNA (Other)	U6		Harris	A panel of eGFP reporters for single base editing by APOBEC-Cas9 editosome complexes. Sci Rep. 2019 Jan 24;9(1):497. doi: 10.1038/s41598-018-36739-9.	gRNA that guides Cas9 and APOBEC complexes to L138 in eGFP. MLM3636
	pAH235	humanized Streptococcus pyogenes Cas9 (Synthetic)	nmt41	S.pombe ura4	Tanaka	Short-Homology-Mediated CRISPR/Cas9-Based Method for Genome Editing in Fission Yeast. G3 (Bethesda). 2019 Feb 12. pii: g3.118.200976. doi: 10.1534/g3.118.200976.	Cas9 expression controlled by the nmt41 promoter pSLF273
	pAH237	gRNA cassette (Schizosaccharomyces pombe), humanized Streptococcus pyogenes Cas9 (Schizosaccharomyces pombe)	S.pombe rrk1, nmt41	LEU2	Tanaka	Short-Homology-Mediated CRISPR/Cas9-Based Method for Genome Editing in Fission Yeast. G3 (Bethesda). 2019 Feb 12. pii: g3.118.200976. doi: 10.1534/g3.118.200976.	Expression of both nmt41p-cas9 and rrk1p-gRNA (LEU2 marker) for CRISPR genome editing in fission yeast pAH233
	pAH243	humanized Streptococcus pyogenes Cas9 (Schizosaccharomyces pombe), gRNA expression module (Schizosaccharomyces pombe)	nmt41, S.pombe rrk1	S.pombe ura4	Tanaka	Short-Homology-Mediated CRISPR/Cas9-Based Method for Genome Editing in Fission Yeast. G3 (Bethesda). 2019 Feb 12. pii: g3.118.200976. doi: 10.1534/g3.118.200976.	Expression of both nmt41p-cas9 and rrk1p-gRNA (ura4 marker) for CRISPR genome editing in fission yeast pSLF273
	pUCC001	HH-BsaI (Synthetic)	TDH3 promoter from S.cerevisiae (already present in backbone)	Hygromycin	Morrissey	Biological Parts for Kluyveromyces marxianus Synthetic Biology Front. Bioeng. Biotechnol., 07 May 2019	Multi-species CRISPR/Cas9 coexpression system, optimized for Golden Gate cloning of gRNA targets pUDP002
	pSDMA65	Hygromycin (HYG) (Synthetic)	ACT1	Hygromycin	Fraser	Targeted Genome Editing via CRISPR in the Pathogen Cryptococcus neoformans. (unpublished)	Codon optimised Streptococcus pyogenes CAS9 ORF regulated by the Cryptococcus neoformans TEF1 promoter and terminator. pBlueScript II SK(-)
	JME4390			LEU2	Rossignol	A set of Yarrowia lipolytica CRISPR/Cas9 vectors for exploiting wild-type strain diversity. Biotechnol Lett. 2020 Jan 23. pii: 10.1007/s10529-020-02805-4. doi: 10.1007/s10529-020-02805-4.	LEU2ex_CrisprCas9-yl_RFP: CAS9 vector with LEU2ex marker for gRNA cloning using GoldenGate pSB1A3
	JME4393			LYS5	Rossignol	A set of Yarrowia lipolytica CRISPR/Cas9 vectors for exploiting wild-type strain diversity. Biotechnol Lett. 2020 Jan 23. pii: 10.1007/s10529-020-02805-4. doi: 10.1007/s10529-020-02805-4.	LYS5ex_CrisprCas9-yl_RFP: CAS9 vector with LYS5ex marker for gRNA cloning using GoldenGate pSB1A3
	JME4472			URA3	Rossignol	A set of Yarrowia lipolytica CRISPR/Cas9 vectors for exploiting wild-type strain diversity. Biotechnol Lett. 2020 Jan 23. pii: 10.1007/s10529-020-02805-4. doi: 10.1007/s10529-020-02805-4.	URA3ex_CrisprCas9-yl_RFP: CAS9 vector with URA3ex marker for gRNA cloning using GoldenGate pSB1A3
	JME4580			Hygromycin	Rossignol	A set of Yarrowia lipolytica CRISPR/Cas9 vectors for exploiting wild-type strain diversity. Biotechnol Lett. 2020 Jan 23. pii: 10.1007/s10529-020-02805-4. doi: 10.1007/s10529-020-02805-4.	HPHex_CrisprCas9-yl_RFP: CAS9 vector with Hygromycinex marker for gRNA cloning using GoldenGate pSB1A3
	JME4599			Nourseothricin	Rossignol	A set of Yarrowia lipolytica CRISPR/Cas9 vectors for exploiting wild-type strain diversity. Biotechnol Lett. 2020 Jan 23. pii: 10.1007/s10529-020-02805-4. doi: 10.1007/s10529-020-02805-4.	NATex_CrisprCas9-yl_RFP: CAS9 vector with Nourseothricin ex marker for gRNA cloning using GoldenGate pSB1A3

Base Edit

Catalytically dead dCas9 fused to a cytidine deaminase protein becomes a specific cytosine base editor that can alter DNA bases without inducing a DNA break. Cytosine base editors convert C->T (or G->A on the opposite strand) within a small editing window specified by the gRNA. Adenine base editors convert adenine to inosine, which is replaced by guanosine to create A->G (or T->C on the opposite strand) mutations.

	Plasmid	Gene/Insert	Promoter	Selectable Marker	PI	Publication	Hidden Extra Search Info
	pRS315e_pGal-dCas9-SH3-pGal-PmCDA1-SHL	SpCas9 (Other), PmCDA1 (Other)	pGal1, pGal10	LEU2	Kondo	Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science. 2016 Aug 4. pii: aaf8729.	Expresses dCas9-SH3 and PmCDA1-SHL in yeast cells pRS315
	pRS315e_pGal-dCas9-PmCDA1	SpCas9 (Other)	pGal1	LEU2	Kondo	Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science. 2016 Aug 4. pii: aaf8729.	Expresses dCas9-PmCDA1 in yeast cells pRS315
	pRS315e_pGal-nCas9(D10A)-PmCDA1	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

Nick

CRISPR/Cas nickase mutants introduce gRNA-targeted single-strand breaks in DNA instead of the double-strand breaks created by wild type Cas enzymes. To use a nickase mutant, you will need two gRNAs that target opposite strands of your DNA in close proximity. These double nicks create a double-strand break (DSB) that is repaired using error-prone non-homologous end joining (NHEJ). Double nicking strategies reduce unwanted off-target effects. Nickase mutants can also be used with a repair template to introduce specific edits via homology-directed repair (HDR).

	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

Activate

Catalytically dead dCas9 fused to a transcriptional activator peptide can increase transcription of a specific gene. Design your gRNA sequence to direct the dCas9-activator to promoter or regulatory regions of your gene of interest. 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 to your specific locus.

	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
	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
	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
	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
	dCas9v2	dCas9-VPR (Saccharomyces cerevisiae)		URA3	Nielsen	Multiplexed CRISPR/Cas9 Genome Editing and Gene Regulation using Csy4 in Saccharomyces cerevisiae. ACS Synth Biol. 2017 Nov 21. doi: 10.1021/acssynbio.7b00259.	TetR inducible dCas9-VPR plasmid with pRPR-NotI-tRPR1 Csy4-ready site pDTU-113
	pCRISPRa_VPR_yl	Codon optimized dCas9-VPR (Synthetic), sgRNA expression cassette (Synthetic)	UAS1B8-TEF(136), SCR1'-tRNA	LEU2	Wheeldon	Multiplexed CRISPR activation of cryptic sugar metabolism enables Yarrowia lipolytica growth on cellobiose. Biotechnol J. 2018 May 5:e1700584. doi: 10.1002/biot.201700584.	CRISPR-dCas9-VPR vector for Yarrowia lipolytica, expressing dCas9-VPR and AvrII site for sgRNA insertion pUC19

Interfere

Catalytically dead dCas9, or dCas9 fused to a transcriptional repressor peptide like KRAB, can knock down gene expression by interfering with transcription. Design your gRNA to target your gene of interest’s promoter/enhancer or the beginning of the coding sequence. If the plasmid you’re using does not also express a gRNA, you will need to use a separate gRNA expression plasmid to target the dCas9-repressor to your specific locus.

	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
	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

Purify

A catalytically inactive Cas9 (dCas9) can be used to purify a region of genomic DNA and its associated proteins, RNA, and DNA. The enCHIP system uses an anti-FLAG antibody to immunoprecipitate FLAG-tagged Cas9. Design your gRNA sequence to direct dCas9 to a specific locus, avoiding known transcription factor and other protein binding sites.

	Plasmid	Gene/Insert	Promoter	Selectable Marker	PI	Publication	Hidden Extra Search Info
	3xFLAG-dCas9/pTEF1p-CYC1t	3xFLAG-dCas9 (Synthetic)	TEF1 promoter	TRP1	Fujii	An enChIP system for the analysis of bacterial genome functions. BMC Res Notes. 2018 Jun 14;11(1):387. doi: 10.1186/s13104-018-3486-3.	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 CRISPR, you will need to express both a Cas protein and a target-specific gRNA in the same cell at the same time. Single plasmids containing both the gRNA and Cas protein act as all-in-one vectors, but their function is often limited to a single category (cut, nick, etc.) On the other hand, gRNA plasmids that do not co-express a Cas protein can be paired with a wide variety of Cas-containing plasmids.

	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