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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 3.

Figure 3

Design of the sgRNAs. (a) The sgRNA is a chimera and consists of three regions: a 20–25-nt-long base-pairing region for specific DNA binding, a 42-nt-long dCas9 handle hairpin for Cas9 protein binding and a 40-nt-long transcription terminator hairpin derived from S. pyogenes. Transcription of sgRNAs should start precisely at its 5′ end. The 12-nt seed region is shaded in orange. (b) The schemes for designing sgRNAs to target the template (T) or nontemplate (NT) DNA strands. When targeting the template DNA strand, the base-pairing region of the sgRNA has the same sequence identity as the transcribed sequence. When targeting the nontemplate DNA strand, the base-pairing region of the sgRNA is the reverse-complement of the transcribed sequence.