CRISPR Plasmids: Zebrafish

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

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	Vector Type	Promoter	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
	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
	pST1374-NLS-flag-linker-Cas9	Human codon optimized Cas9	Mammalian Expression, CRISPR	CMV	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)
	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
	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
	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+
	pCS2+hSpCas9	humanized S.pyogenes Cas9	Mammalian Expression, CRISPR ; medaka, zebrafish	SP6	Kinoshita	Targeted mutagenesis using CRISPR/Cas system in medaka. Biol Open. 2014 Apr 11;3(5):362-71. doi: 10.1242/bio.20148177.	Encode hSpCas9 for in vitro transcription with Sp6 pCS2
	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
	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)
	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)
	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
	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
	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
	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)
	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)
	pT3TS-zAsCpf1	AsCpf1 (Danio rerio)	CRISPR		Giraldez	CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing. Nat Commun. 2017 Dec 8;8(1):2024. doi: 10.1038/s41467-017-01836-2.	plasmid to carry out IVT of AsCpf1 (zebrafish codon optimized) pT3TS
	pME-cas9-2A-mCherry	cas9-2A-mCherry (Danio rerio)	CRISPR		Deng	Neutrophil-specific knockout demonstrates a role for mitochondria in regulating neutrophil motility in zebrafish. Dis Model Mech. 2018 Mar 28;11(3). pii: dmm.033027. doi: 10.1242/dmm.033027.	An entry vector with cas9 -2A-mCherry pME
	pTol2-hsp70l:Cas9-t2A-GFP, 5xU6:sgRNA	5xU6:sgRNA (Danio rerio), Cas9-t2A-GFP (Danio rerio)	CRISPR	U6, hsp70	Schier	Simultaneous single-cell profiling of lineages and cell types in the vertebrate brain. Nat Biotechnol. 2018 Mar 28. pii: nbt.4103. doi: 10.1038/nbt.4103.	Expression of heat shock inducible Cas9-GFP and U6-driven 5 individual gRNAs for scGESTALT in zebrafish Tol2
	pME-nCas9n-P2A-mKate2-pA	nCas9n and mKate2 (Synthetic)	Zebrafish plasmids		Parton	In vivo cell biological screening identifies an endocytic capture mechanism for T-tubule formation. Nat Commun. 2020 Jul 24;11(1):3711. doi: 10.1038/s41467-020-17486-w.	Multisite gateway vector for expression of nCas9n with bicistronic expression of mKate2 pDONR 221
	MiniCoopR U6:gRNA, mitfa:Cas9		CRISPR ; Tol2		Zon	Human tumor genomics and zebrafish modeling identify SPRED1 loss as a driver of mucosal melanoma. Science. 2018 Nov 1. pii: science.aau6509. doi: 10.1126/science.aau6509.	Targets a gene and expresses zebrafish mitfa specifically in zebrafish melanocytes MiniCoopR
	MiniCoopR 2xU6:gRNA, mitfa:Cas9		CRISPR ; Tol2		Zon	Human tumor genomics and zebrafish modeling identify SPRED1 loss as a driver of mucosal melanoma. Science. 2018 Nov 1. pii: science.aau6509. doi: 10.1126/science.aau6509.	Targets 2 genes and expresses zebrafish mitfa specifically in zebrafish melanocytes MiniCoopR
	pT3TS-nCas9n-A71	nCas9n (Other)	RNA in vitro transcription vector	T3	Berman	Genome editing in zebrafish using high-fidelity Cas9 nucleases (unpublished)	micro-injection of poly-adenylated nCas9n RNA pT3TS
	pT3TS-Hypa-nCas9n-A69	Hypa-nCas9n (Other)	RNA in vitro transcription vector	T3	Berman	Genome editing in zebrafish using high-fidelity Cas9 nucleases (unpublished)	micro-injection of poly-adenylated Hypa-nCas9n RNA pT3TS
	pT3TS-HiFi-nCas9n-A72	HiFi-nCas9n (Other)	RNA in vitro transcription vector	T3	Berman	Genome editing in zebrafish using high-fidelity Cas9 nucleases (unpublished)	micro-injection of poly-adenylated HiFi-nCas9n RNA pT3TS
	pTol2 hsp70l loxP-DsRed-loxP-nCas9n-GFP	hsp70l promoter (Danio rerio), floxed DsRed cassette (Synthetic), Cas9-GFP (Synthetic)	Cre/Lox, CRISPR		Brand	Cre-Controlled CRISPR mutagenesis provides fast and easy conditional gene inactivation in zebrafish. Nat Commun. 2021 Feb 18;12(1):1125. doi: 10.1038/s41467-021-21427-6.	Cre-dependet Cas9-GFP expression pBluescript
	pTol2 hsp70l loxP-Non-FP-loxP-nCas9n-GFP	hsp70l promoter (Danio rerio), floxed Non-FP cassette (Synthetic), Cas9-GFP (Synthetic)	Cre/Lox, CRISPR		Brand	Cell-fate plasticity, adhesion and cell sorting complementarily establish a sharp midbrain-hindbrain boundary. Development. 2020 Jun 8;147(11). pii: dev.186882. doi: 10.1242/dev.186882.	Cre-dependet Cas9-GFP expression pBluescript
	OmEF1aCas9P2ApuroSB	Zebrafish optimized Cas9 (nls-zcas9-nls from Wenbiao Chen Lab plasmid pCS2-nCas9n Addgene #47929) (Synthetic)	CRISPR ; Transposon	Oreochromis mossambicus EF1 alpha promoter (OmEF1a)	Kueltz	An efficient vector-based CRISPR/Cas9 system in an Oreochromis mossambicus cell line using endogenous promoters. Sci Rep. 2021 Apr 12;11(1):7854. doi: 10.1038/s41598-021-87068-3.	Stable expression of a zebrafish optimized Cas9 driven by a tilapia promoter in fish cells pSBbi-GP

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)	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