CRISPR Plasmids: Yeast

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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.

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