CRISPR/Cas9 plasmids for use in Zebrafish

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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 zebrafish.

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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	Vector Type	Promoter	Selectable Marker	PI	Publication	Hidden Extra Search Info
	MLM3613	Streptococcus pyogenes Cas9	CRISPR ; zebrafish expression	CMV		Joung	Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol. 2013 Jan 29. doi: 10.1038/nbt.2501.	Expresses Cas9 nuclease (Streptococcus pyogenes) from CMV and T7 promoters unknown	Add to Cart
	MLM3639	Streptococcus pyogenes Cas9-3X Flag	CRISPR ; zebrafish expression	CMV		Joung	Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol. 2013 Jan 29. doi: 10.1038/nbt.2501.	unknown	Add to Cart
	pST1374-NLS-flag-linker-Cas9	Human codon optimized Cas9	Mammalian Expression, CRISPR	CMV	Blasticidin	Huang	Generation of gene-modified mice via Cas9/RNA-mediated gene targeting. Cell Res. 2013 Apr 2. doi: 10.1038/cr.2013.46.	pST1374 (Addgene #13426)	Add to Cart
	pT3TS-nCas9n	Cas9 (Synthetic)	Bacterial Expression, CRISPR	T3		Chen	Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proc Natl Acad Sci U S A. 2013 Aug 5.	pT3T7	Add to Cart
	pCS2-Cas9	Cas9 (Other)	Mammalian Expression, CRISPR ; xenopus and zebrafish expression	SP6		Schier	Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs. PLoS One. 2014 May 29;9(5):e98186. doi: 10.1371/journal.pone.0098186. eCollection 2014.	For in vivo expression of Cas9 or generation of Cas9 mRNA pCS2	Add to Cart
	pCS2-nCas9n	nls-zcas9-nls (Synthetic)	Mammalian Expression, CRISPR	CMV		Chen	Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proc Natl Acad Sci U S A. 2013 Aug 5.	expression of an optimized Cas9 for genome-editing in zebrafish pCS2+	Add to Cart
	pCS2+hSpCas9	humanized S.pyogenes Cas9	Mammalian Expression, CRISPR ; medaka, zebrafish	SP6		Kinoshita	Kinoshita lab Cas9 plasmids (unpublished)	Encode hSpCas9 for in vitro transcription with Sp6 pCS2	Add to Cart
	pCS2-nCas9n-nanos 3’UTR	nanos 3' UTR (Danio rerio)	CRISPR	SP6		Giraldez	CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo. Nat Methods. 2015 Aug 31. doi: 10.1038/nmeth.3543.	expression of a germ-line targeted, codon optimized Cas9 for genome-editing in zebrafish pCS2-nCas9n nanos3 cb725, nanos, nanos1, nos1	Add to Cart
	pME-Cas9	Cas9 (Danio rerio)	CRISPR ; Tol2 Middle Entry vector for Gateway			Zon	A CRISPR/Cas9 Vector System for Tissue-Specific Gene Disruption in Zebrafish. Dev Cell. 2015 Mar 4. pii: S1534-5807(15)00075-1. doi: 10.1016/j.devcel.2015.01.032.	Middle Entry clone for Gateway containing a zebrafish codon-optimized Cas9 flanked by 2 NLS pME-MCS (Tol2Kit #237)	Add to Cart
	pME-Cas9-T2A-GFP	Cas9-T2A-GFP (Danio rerio)	CRISPR ; Tol2 Middle Entry vector for Gateway			Zon	A CRISPR/Cas9 Vector System for Tissue-Specific Gene Disruption in Zebrafish. Dev Cell. 2015 Mar 4. pii: S1534-5807(15)00075-1. doi: 10.1016/j.devcel.2015.01.032.	Middle Entry clone for Gateway containing a zebrafish codon-optimized Cas9 flanked by 2 NLS and followed by inframe GFP. Cas9 and GFP are separated by the sequence of a T2A self-cleaving peptide. pME-MCS (Tol2Kit #237)	Add to Cart
	pME-Cas9	codon optimized Cas9 (Other)	CRISPR			Chen	Multiplex Conditional Mutagenesis Using Transgenic Expression of Cas9 and sgRNAs. Genetics. 2015 Apr 8. pii: genetics.115.176917.	expresses codon optimized Cas9 in zebrafish pME-MCS	Add to Cart
	pUAS:Cas9T2AGFP;U6:sgRNA1;U6:sgRNA2	5x UAS (Saccharomyces cerevisiae), Cas9 (Other), T2A, GFP, U6 promoter (Danio rerio)	CRISPR			Del Bene	2C-Cas9: a versatile tool for clonal analysis of gene function. Genome Res. 2016 Mar 8.	Tissue-specific knock-out in zebrafish, Cas9 and GFP driven by a UAS promoter pminiTol2	Add to Cart
	pUAS:Cas9T2ACre;U6:sgRNA1;U6:sgRNA2	5xUAS, Cas9, Cre, U6 promoter	CRISPR			Del Bene	2C-Cas9: a versatile tool for clonal analysis of gene function. Genome Res. 2016 Mar 8.	Tissue-specific knock-out in zebrafish, Cas9 and Cre driven by a UAS promoter pminiTol2	Add to Cart
	pMJ922	SpCas9 (Other)	Bacterial Expression	T7		Jinek	Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes. Development. 2016 Apr 29. pii: dev.134809.	Expression of His6-MBP-tagged Cas9-NLS-EGFP protein in E. coli pET His6 MBP N10 TEV LIC cloning vector (2C-T)	Add to Cart
	pMJ923	SpCas9 (Other)	Bacterial Expression	T7		Jinek	Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes. Development. 2016 Apr 29. pii: dev.134809.	Expression of His6-MBP-tagged Cas9-NLS-mCherry protein in E. coli pET His6 MBP N10 TEV LIC cloning vector (2C-T)	Add to Cart

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)	Delivery	Resistance	Co-expressed Cas9	Depositing lab
Cas9 species = S. pyogenes (PAM = NGG)
	pT7-gRNA	T7	BsmBI	In vitro transcription		none, need Cas9 plasmid	Chen and Wente
	DR274	T7	BsaI	In vitro transcription		none, need Cas9 plasmid	Joung

Last Updated: February 28, 2014