CRISPR-Cas/RGN expression plasmids for Zebrafish

This page lists plasmids required to practice CRISPR-Cas/RNA-Guided Nuclease (RGN) technology in zebrafish. The Joung Lab has demonstrated the use of these vectors to efficiently modify nine different target sites in endogenous zebrafish genes (Hwang & Fu et al., Nat Biotechnol. 2013).

The Cas9 expression vector listed below (pMLM3613) harbors a T7 promoter that drives expression of a transcript encoding the Cas9 endonuclease. Details for producing mRNA suitable for expression of Cas9 in zebrafish can be found in Hwang & Fu et al., Nat Biotechnol. 2013.

The guide RNA (gRNA) expression vector listed below (pDR274) can be used to construct customized gRNAs targeted to a sequence of interest. Potential target sites can be identified using the publicly available, web-based ZiFiT Targeter software. Specific gRNA expression plasmids can be built by cloning a pair of annealed oligonucleotides (each 26 nts in length) into BsaI-digested pDR274 vector. The oligonucleotides to be cloned can be designed using the ZiFiT Targeter software http://zifit.partners.org/ZiFiT/ChoiceMenu.aspx by choosing the “CRISPR/Cas: Design oligos for making guide RNAs” link under the “Support Tools” heading. Completed vectors express a customized guide RNA from a T7 promoter.

Individual plasmids can be ordered via the links below:

ID	Plasmid
42251	MLM3613 (Cas9 expression vector)
42250	DR274 (guide RNA expression vector)