CRISPR-Cas/RGN expression plasmids for human cells

This page lists plasmids required to practice CRISPR-Cas/RNA-Guided Nuclease (RGN) technology in human cells.

The Cas9 expression vector listed below (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 listed below (MLM3636) 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 BsmBI-digested MLM3636 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 U6 promoter.

Individual plasmids can be ordered via the links below:

ID	Plasmid
43860	MLM3636 (human gRNA expression vector)
42252	MLM3639 (Cas9 expression vector with C-term 3X FLAG tag)
43861	JDS246 (mammalian codon-optimized Cas9 with C-term 3X FLAG tag)