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CRISPR header icon CRISPR/Cas9 plasmids for use in Zebrafish


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.

Cut

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  
MLM3613Streptococcus pyogenes Cas9CRISPR ; zebrafish expressionCMV 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
MLM3639Streptococcus pyogenes Cas9-3X FlagCRISPR ; zebrafish expressionCMV 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-Cas9Human codon optimized Cas9Mammalian Expression, CRISPRCMVBlasticidin 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-nCas9nCas9 (Synthetic)Bacterial Expression, CRISPRT3 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-Cas9Cas9 (Other)Mammalian Expression, CRISPR ; xenopus and zebrafish expressionSP6 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-nCas9nnls-zcas9-nls (Synthetic)Mammalian Expression, CRISPRCMV 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+hSpCas9humanized S.pyogenes Cas9Mammalian Expression, CRISPR ; medaka, zebrafishSP6 Kinoshita Kinoshita lab Cas9 plasmids (unpublished) Encode hSpCas9 for in vitro transcription with Sp6 pCS2 Add to Cart
pCS2-nCas9n-nanos 3’UTRnanos 3' UTR (Danio rerio)CRISPRSP6 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-Cas9Cas9 (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-GFPCas9-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-Cas9codon 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:sgRNA25x 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:sgRNA25xUAS, Cas9, Cre, U6 promoterCRISPR 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
pMJ922SpCas9 (Other)Bacterial ExpressionT7 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
pMJ923SpCas9 (Other)Bacterial ExpressionT7 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

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