CRISPR Plasmids: Visualize Genomic Loci

Using catalytically inactive Cas9 (dCas9) fused to a fluorescent marker like GFP, researchers have turned dCas9 into a customizable DNA labeler compatible with fluorescence microscopy in living cells. Alternatively, gRNAs can be fused to protein-interacting RNA aptamers, which recruit specific RNA-binding proteins (RBPs) tagged with fluorescent proteins to visualize targeted genomic loci.

CRISPR imaging has numerous advantages over other imaging techniques, including that it is easy to implement due to the simplicity of gRNA design, programmable for different genomic loci, capable of detecting multiple genomic loci, and compatible with live cell imaging. Compared to techniques like fluorescence in situ hybridization (FISH), CRISPR imaging offers a unique method for detecting the chromatin dynamics in living cells.

The fluorescent CRISPR system has been used for dynamic tracking of repetitive and non-repetitive genomic loci, as well as chromosome painting in living cells. Visualizing a specific genomic locus requires recruitment of many copies of labeled proteins to the given region. For example, chromosome-specific repetitive loci can be efficiently visualized in living cells using a single gRNA that has multiple targeted sequences in close proximity. A non-repetitive genomic locus can also be labeled by co-delivering multiple gRNAs that tile the locus. Chromosome painting requires delivery of hundreds of gRNAs with target sites throughout the chromosome.

Mammalian

	Plasmid	Gene/Insert	Promoter	Selectable Marker	PI	Publication	Hidden Extra Search Info
	pHR-SFFV-dCas9-BFP	dCas9-BFP fusion (Homo sapiens)	SFFV		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.	Human expression vector containing SFFV promoter, dCas9 that is fused to 2x NLS and tagBFP pHR
	pSLQ1658-dCas9-EGFP	dCas9 fuse to EGFP (Homo sapiens)	MSCV LTR promoter	Puromycin	Qi	Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System. Cell. 2013 Dec 19;155(7):1479-91. doi: 10.1016/j.cell.2013.12.001.	Template for NLS-dCas9-NLS-EGFP fusion protein for CRISPR imaging (the recipient vector can be TetON 3G promoter system) pMSCVpuro
	pBSKΔB-TRE-dCas9-EGFP	dCas9-EGFP (Synthetic)	TRE		Ochiai	Simultaneous live imaging of the transcription and nuclear position of specific genes. Nucleic Acids Res. 2015 Jun 19. pii: gkv624.	Plasmid for tet-inducible expression of dCas9-EGFP in mammalian cells pBlueScript II SK (+)
	pHAGE-EFS-dCas9-GFP	Sp dCas9 (Synthetic)	EFS		Pederson	Multicolor CRISPR labeling of chromosomal loci in human cells. Proc Natl Acad Sci U S A. 2015 Mar 10;112(10):3002-7. doi: 10.1073/pnas.1420024112. Epub 2015 Feb 23.	Expression of Sp dCas9-GFP in mammalian cells pHAGE-DEST
	pHAGE-SSFV-dCas9-GFP	Sp dCas9 (Synthetic)	SSFV		Pederson	Multicolor CRISPR labeling of chromosomal loci in human cells. Proc Natl Acad Sci U S A. 2015 Mar 10;112(10):3002-7. doi: 10.1073/pnas.1420024112. Epub 2015 Feb 23.	Expression of Sp dCas9-GFP in mammalian cells pHAGE-DEST
	pHAGE-TO-dCas9-GFP	Sp dCas9 (Synthetic)	CMV-TO		Pederson	Multicolor CRISPR labeling of chromosomal loci in human cells. Proc Natl Acad Sci U S A. 2015 Mar 10;112(10):3002-7. doi: 10.1073/pnas.1420024112. Epub 2015 Feb 23.	Expression of Sp dCas9-GFP in mammalian cells pHAGE-DEST
	pHAGE-TO-dCas9-3XGFP	Sp dCas9 (Synthetic)	CMV-TO	GFP	Pederson	Multicolor CRISPR labeling of chromosomal loci in human cells. Proc Natl Acad Sci U S A. 2015 Mar 10;112(10):3002-7. doi: 10.1073/pnas.1420024112. Epub 2015 Feb 23.	Expression of Sp dCas9-3XGFP in mammalian cells pHAGE-DEST
	pHAGE-TO-dCas9-3XmCherry	Sp dCas9 (Synthetic)	CMV-TO	mCherry	Pederson	Multicolor CRISPR labeling of chromosomal loci in human cells. Proc Natl Acad Sci U S A. 2015 Mar 10;112(10):3002-7. doi: 10.1073/pnas.1420024112. Epub 2015 Feb 23.	Expression of Sp dCas9-3XmCherry in mammalian cells pHAGE-DEST
	pHAGE-TO-nmdCas9-3XGFP	Nm dCas9 (Synthetic)	CMV-TO		Pederson	Multicolor CRISPR labeling of chromosomal loci in human cells. Proc Natl Acad Sci U S A. 2015 Mar 10;112(10):3002-7. doi: 10.1073/pnas.1420024112. Epub 2015 Feb 23.	Expression of Nm dCas9-3XGFP in mammalian cells pHAGE-DEST
	pHAGE-TO-nmdCas9-3XmCherry	Nm dCas9 (Synthetic)	CMV-TO		Pederson	Multicolor CRISPR labeling of chromosomal loci in human cells. Proc Natl Acad Sci U S A. 2015 Mar 10;112(10):3002-7. doi: 10.1073/pnas.1420024112. Epub 2015 Feb 23.	Expression of Nm dCas9-3XmCherry in mammalian cells pHAGE-DEST
	pHAGE-TO-nls-st1dCas9-3nls-3XGFP	St1 dCas9 (Synthetic)	CMV-TO		Pederson	Multicolor CRISPR labeling of chromosomal loci in human cells. Proc Natl Acad Sci U S A. 2015 Mar 10;112(10):3002-7. doi: 10.1073/pnas.1420024112. Epub 2015 Feb 23.	Expression of St1 dCas9-3XGFP in mammalian cells pHAGE-DEST
	pHAGE-TO-nls-st1dCas9-3nls-3XGFP-nls	St1 dCas9 (Synthetic)	CMV-TO		Pederson	Multicolor CRISPR labeling of chromosomal loci in human cells. Proc Natl Acad Sci U S A. 2015 Mar 10;112(10):3002-7. doi: 10.1073/pnas.1420024112. Epub 2015 Feb 23.	Expression of St1 dCas9-3XGFP in mammalian cells pHAGE-DEST
	pHAGE-TO-nls-st1dCas9-3nls-3XGFP-2nls	St1 dCas9 (Synthetic)	CMV-TO		Pederson	Multicolor CRISPR labeling of chromosomal loci in human cells. Proc Natl Acad Sci U S A. 2015 Mar 10;112(10):3002-7. doi: 10.1073/pnas.1420024112. Epub 2015 Feb 23.	Expression of St1 dCas9-3XGFP in mammalian cells pHAGE-DEST
	pHAGE-TO-nls-st1dCas9-3nls-3XTagBFP2	St1 dCas9 (Synthetic)	CMV-TO		Pederson	Multicolor CRISPR labeling of chromosomal loci in human cells. Proc Natl Acad Sci U S A. 2015 Mar 10;112(10):3002-7. doi: 10.1073/pnas.1420024112. Epub 2015 Feb 23.	Expression of St1 dCas9-3XTagBFP2 in mammalian cells pHAGE-DEST
	pCDNA3.1- dCas9-2xNLS-EGFP	dCas9-2xNLS-EGFP	CMV		Yeo	Programmable RNA Tracking in Live Cells with CRISPR/Cas9. Cell. 2016 Mar 16. pii: S0092-8674(16)30204-5. doi: 10.1016/j.cell.2016.02.054.	Expresses dCas9-2xNLS-EGFP in mammalian cells pCDNA3.1(-)
	pHAGE-TO-MCP-3XBFPnls	MCP (Synthetic)	CMV-TO		Pederson	Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow. Nat Biotechnol. 2016 Apr 18. doi: 10.1038/nbt.3526.	MCP-3XBFPnls pHAGE-DEST
	pHAGE-EFS-MCP-3XBFPnls	MCP (Synthetic)	EFS		Pederson	Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow. Nat Biotechnol. 2016 Apr 18. doi: 10.1038/nbt.3526.	MCP-3XBFPnls pHAGE-DEST
	pHAGE-EFS-PCP-3XGFPnls	PCP (Synthetic)	EFS		Pederson	Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow. Nat Biotechnol. 2016 Apr 18. doi: 10.1038/nbt.3526.	PCP-3XGFPnls pHAGE-DEST
	pHAGE-EFS-PCP-3XNeon-nls	PCP (Synthetic)	EFS		Pederson	Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow. Nat Biotechnol. 2016 Apr 18. doi: 10.1038/nbt.3526.	PCP-3XNeon-nls pHAGE-DEST
	pHAGE-EFS-N22p-3XRFPnls	N22p (Synthetic)	EFS		Pederson	Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow. Nat Biotechnol. 2016 Apr 18. doi: 10.1038/nbt.3526.	N22p-3XRFPnls pHAGE-DEST
	pLH-sgRNA1-2XMS2	sgRNA1-2XMS2 (Synthetic)	U6	Hygromycin	Pederson	Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow. Nat Biotechnol. 2016 Apr 18. doi: 10.1038/nbt.3526.	sgRNA1-2XMS2 pLKO.1
	pLH-sgRNA1-2XPP7	sgRNA1-2XPP7 (Synthetic)	U6	Hygromycin	Pederson	Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow. Nat Biotechnol. 2016 Apr 18. doi: 10.1038/nbt.3526.	sgRNA1-2XPP7 pLKO.1
	pLH-sgRNA1-2XboxB	sgRNA1-2XboxB (Synthetic)	U6	Hygromycin	Pederson	Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow. Nat Biotechnol. 2016 Apr 18. doi: 10.1038/nbt.3526.	sgRNA1-2XboxB pLKO.1
	pLH-sgRNA1-MS2-PP7	sgRNA1-2XMS2-PP7 (Synthetic)	U6	Hygromycin	Pederson	Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow. Nat Biotechnol. 2016 Apr 18. doi: 10.1038/nbt.3526.	sgRNA1-MS2-PP7 pLKO.1
	pLH-sgRNA1-PP7-boxB	sgRNA1-2XPP7-boxB (Synthetic)	U6		Pederson	Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow. Nat Biotechnol. 2016 Apr 18. doi: 10.1038/nbt.3526.	sgRNA1-PP7-boxB pLKO.1
	pLH-sgRNA1-boxB-MS2	sgRNA1-boxB-MS2 (Synthetic)	U6	Hygromycin	Pederson	Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow. Nat Biotechnol. 2016 Apr 18. doi: 10.1038/nbt.3526.	sgRNA1-boxB-MS2 pLKO.1
	pLH-sgRNA1-boxB-MS2-PP7	sgRNA1-boxB-MS2-PP7 (Synthetic)	U6	Hygromycin	Pederson	Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow. Nat Biotechnol. 2016 Apr 18. doi: 10.1038/nbt.3526.	sgRNA1-boxB-MS2-PP7 pLKO.1
	pHAGE-TO-dCas9-P2A-HSA	dCas9-P2A-HSA (Synthetic)	CMV-TO		Pederson	CRISPR-Sirius: RNA scaffolds for signal amplification in genome imaging. Nat Methods. 2018 Nov;15(11):928-931. doi: 10.1038/s41592-018-0174-0. Epub 2018 Oct 30.	CRISPR-Sirius plasmid pHAGE-TO
	pHAGE-EFS-MCP-HALOnls	MCP-HALOnls (Synthetic)	EFS		Pederson	CRISPR-Sirius: RNA scaffolds for signal amplification in genome imaging. Nat Methods. 2018 Nov;15(11):928-931. doi: 10.1038/s41592-018-0174-0. Epub 2018 Oct 30.	CRISPR-Sirius plasmid pHAGE-EFS
	pHAGE-EFS-PCP-GFPnls	PCP-GFPnls (Synthetic)	EFS		Pederson	CRISPR-Sirius: RNA scaffolds for signal amplification in genome imaging. Nat Methods. 2018 Nov;15(11):928-931. doi: 10.1038/s41592-018-0174-0. Epub 2018 Oct 30.	CRISPR-Sirius plasmid pHAGE-EFS
	pLH-sgRNA-Sirius-8XMS2	sgRNA-Sirius-8XMS2 (Synthetic)	human U6 promoter	Hygromycin	Pederson	CRISPR-Sirius: RNA scaffolds for signal amplification in genome imaging. Nat Methods. 2018 Nov;15(11):928-931. doi: 10.1038/s41592-018-0174-0. Epub 2018 Oct 30.	CRISPR-Sirius plasmid pLKO.1
	pLH-sgRNA-Sirius-8XPP7	sgRNA-Sirius-8XPP7 (Synthetic)	human U6 promoter	Hygromycin	Pederson	CRISPR-Sirius: RNA scaffolds for signal amplification in genome imaging. Nat Methods. 2018 Nov;15(11):928-931. doi: 10.1038/s41592-018-0174-0. Epub 2018 Oct 30.	CRISPR-Sirius plasmid pLKO.1
	pLH-sgRNA-Sirius-4X(MS2-PP7)	sgRNA-Sirius-4X(MS2-PP7) (Synthetic)	human U6 promoter	Hygromycin	Pederson	CRISPR-Sirius: RNA scaffolds for signal amplification in genome imaging. Nat Methods. 2018 Nov;15(11):928-931. doi: 10.1038/s41592-018-0174-0. Epub 2018 Oct 30.	CRISPR-Sirius plasmid pLKO.1
	pPUR-hU6-sgRNA-Sirius-8XMS2	sgRNA-Sirius-8XMS2 (Synthetic)	human U6 promoter	Puromycin	Pederson	CRISPR-Sirius: RNA scaffolds for signal amplification in genome imaging. Nat Methods. 2018 Nov;15(11):928-931. doi: 10.1038/s41592-018-0174-0. Epub 2018 Oct 30.	CRISPR-Sirius plasmid pLKO.1
	pPUR-mU6-sgRNA-Sirius-8XPP7	sgRNA-Sirius-8XPP7 (Synthetic)	mouse U6 promoter	Puromycin	Pederson	CRISPR-Sirius: RNA scaffolds for signal amplification in genome imaging. Nat Methods. 2018 Nov;15(11):928-931. doi: 10.1038/s41592-018-0174-0. Epub 2018 Oct 30.	CRISPR-Sirius plasmid pLKO.1
	pPUR-hU6-Sirius-8XMS2-mU6-Sirius-8XPP7	hU6-Sirius-8XMS2-mU6-Sirius-8XPP7 Cassette (Synthetic)	U6 promoters	Puromycin	Pederson	CRISPR-Sirius: RNA scaffolds for signal amplification in genome imaging. Nat Methods. 2018 Nov;15(11):928-931. doi: 10.1038/s41592-018-0174-0. Epub 2018 Oct 30.	CRISPR-Sirius plasmid pLKO.1