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Notes: we have recently discovered that an optimized sgRNA design could dramatically enhance the efficiency of using S. pyogenes Cas9 for genome editing, regulation (3-10 fold increase for activation and repression), and imaging. The new sgRNA sequence is the following:
5'- GN19GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG TGGCACCGAGTCGGTGCTTTTTTT-3’
Where the GN19 (N = A/T/C/G) is the basepairing sequence to the target DNA; the modified basepairs are underlined.
Its structure (predicted using mFold):
Update (Feb 18th, 2014): The plasmids for the CRISPR imaging system is now available at Addgene (http://www.addgene.org/Stanley_Qi/), cat log # 51023, 51024, 51025.
Due to MTA issues, we can’t distribute the original dCas9-EGFP fusion system that is based on the Tet ON 3G expression vector (Clontech). The #51023 contains the full dCas9-EGFP fusion cassette, and can be sub-cloned to a low-expressing vector (CRITICAL!) for live imaging in mammalian cells.