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. Author manuscript; available in PMC: 2014 Nov 1.
Published in final edited form as: Nat Protoc. 2013 Oct 17;8(11):2180–2196. doi: 10.1038/nprot.2013.132

Figure 5.

Figure 5

Cloning strategy for concatenating multiple sgRNA expression cassettes onto the same plasmid. (a) The cloning method for bacterial sgRNA expression. The donor sgRNA vector is digested using EcoRI and BamHI, and the backbone vector is digested using EcoRI and BglII. Ligation of the two fragments recreates the compatible restriction sites (BglII and BamHI), which can be used for the next round of sgRNA insertion. (b) The cloning method for human sgRNA expression. The main difference from a is that the insert is first PCR amplified to introduce a 5′ BglII site for subsequent BioBrick cloning.