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There are two control plasmids, with short names “E” and “F”. Both of these express BFP and GFP. However, “F” has a gRNA against the GFP. Thus if Cas9 works in your system, you will have reduced GFP in the “F” plasmid. “E” is a no-target control to compare with.

For generating new sgRNA plasmids, you will want to subclone from the backbone “bb”. For now, see map bb below. To generate new guides, see ….

For amplification of the library “L”, see https://www.addgene.org/static/data/plasmids/67/67988/67988-attachment_NiqBVnwSwjeM.docx

A long protocol for virus production, T cell purification and culture etc, as we did it, is attached as a supplementary to our Cell paper.

 

Sequences as verified by Addgene (they do whole-plasmid resequencing using NGS):

bb: pMSCV-U6sgRNA(BbsI)-PGKpuro2ABFP

E: pMSCV(W-)U6sgRNA5(BbsI)-PGKpuroBFP

F: pMSCV(gfp)U6sgRNA5(BbsI)-PGKpuroBFP