Keith Joung Lab: Joung lab unpublished CRISPR plasmids
Joung JK
Unpublished
CRISPR-Cas/RNA-Guided Nuclease (RGN) technology in human cells.
The Cas9 expression vectors (MLM3639 and JDS246) harbor a CMV promoter that drives expression of a transcript encoding the Cas9 endonuclease. Both vectors also express a C-terminal 3X FLAG tag. MLM3639 is the same as the Cas9 expression vector used for zebrafish (MLM3613) except for the addition of the 3X FLAG tag. JDS246 has been codon optimized for expression in mammalian cells.
The guide RNA (gRNA) expression vector (MLM3636) 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 nt in length) into BsmBI-digested MLM3636 vector. The oligonucleotides to be cloned can be designed using the ZiFiT Targeter (Link opens in a new window) tool 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 U6 promoter.