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CRISPR Plasmids: Zebrafish


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

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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
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
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
pST1374-NLS-flag-linker-Cas9Human codon optimized Cas9Mammalian Expression, CRISPRCMV 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-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
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
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+
pCS2+hSpCas9humanized S.pyogenes Cas9Mammalian Expression, CRISPR ; medaka, zebrafishSP6 Kinoshita Kinoshita lab Cas9 plasmids (unpublished) Encode hSpCas9 for in vitro transcription with Sp6 pCS2
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
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)
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)
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
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
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
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)
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)

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

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