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. 2015 May 26;43(12):6049–6061. doi: 10.1093/nar/gkv546

Figure 2.

Figure 2.

Generation of unit-sized crRNA by transcription termination in vivo. (A). The pG8_crRNA plasmid designed to express crRNA containing a spacer (shown in blue) targeting the M13 phage and E. coli strains used in this work are schematically presented. Colored arrows represent cas3 (orange), cse1cse2cas7cas5cas6e (yellow) and cas1 and cas2 (dark and light green, respectively). Transcription of cas3 and cse1cse2cas7cas5cas6ecas1cas2 operon is driven by inducible promoters (shown as thin black arrows). Rhombi indicate CRISPR repeats, a blue rectangle in KD263 represents the g8 spacer. The crRNA transcript produced from the pG8_crRNA plasmid is indicated. (B). Northern blot analysis of RNA prepared from cells shown in panel A and hybridizing to g8 spacer probe (lanes 1–5) or from Cascade purified from indicated cells (lanes 6–8). Likely structures of RNAs migrating as separate bands on the gel are schematically shown on the right. Question marks indicate uncertainty about the structure of 5′ ends of crRNA transcripts. T: total RNA, C: RNA extracted from Cascade.