Efficient genome editing in zebrafish using a CRISPR-Cas system.
Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, Peterson RT, Yeh JR, Joung JK
Nat Biotechnol. 2013 Jan 29. doi: 10.1038/nbt.2501.
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PubMed
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Article
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.
The expression plasmids encoding the guide RNAs (gRNAs) used in these experiments are listed below (Addgene plasmid #42241–42249). Note that gRNA produced from each of these vectors were used in conjunction with mRNA produced from the Cas9 expression vector (MLM3613). Additional details can be found in the paper and users are encouraged to post questions to the Genome Engineering newsgroup (which can be joined here (Link opens in a new window)).
The Cas9 expression vector listed below (MLM3613) 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 the publication.
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 ZiFiT Targeter (Link opens in a new window) tool. 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 (Link opens in a new window) 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.
Plasmids from Article
| ID | Plasmid | Purpose |
|---|---|---|
| 42241 | Zebrafish-gRNA-0001 | gRNA targeted to zebrafish gene apoea |
| 42242 | Zebrafish-gRNA-0002 | gRNA targeted to zebrafish gene drd3 |
| 42243 | Zebrafish-gRNA-0003 | gRNA targeted to zebrafish gene fh [site #1] |
| 42244 | Zebrafish-gRNA-0004 | gRNA targeted to zebrafish gene fh [site #2] |
| 42245 | Zebrafish-gRNA-0005 | gRNA targeted to zebrafish gene gsk3b |
| 42246 | Zebrafish-gRNA-0006 | gRNA targeted to zebrafish gene rgs4 |
| 42247 | Zebrafish-gRNA-0007 | gRNA targeted to zebrafish gene th1 |
| 42248 | Zebrafish-gRNA-0008 | gRNA targeted to zebrafish gene tia1l |
| 42249 | Zebrafish-gRNA-0009 | gRNA targeted to zebrafish gene tph1a |
| 42250 | DR274 | guide RNA (gRNA) expression vector used to create a gRNA to a specific sequence; uses T7 promoter for in vitro transcription |
| 42251 | MLM3613 | Expresses Cas9 nuclease (Streptococcus pyogenes) from CMV and T7 promoters |
| 42252 | MLM3639 | Cas9 expression vector |