Skip to main content
. Author manuscript; available in PMC: 2018 Apr 13.
Published in final edited form as: Science. 2008 Aug 15;321(5891):960–964. doi: 10.1126/science.1159689

Fig. 2.

Fig. 2

Cascade cleaves CRISPR RNA precursors into small RNAs of ∼57 nucleotides (marked by arrows). (A) Northern analysis of total RNA of WT E. coli K12 (WT), a non-cas gene knockout (Δu, uidA, β-glucuronidase), and Cascade gene knockouts using the single-stranded spacer sequence BG2349 (table S2) as a probe. (B) Northern blot as in (A) of total RNA from E. coli BL21 (DE3) expressing the E. coli K12 pre-crRNA and either the complete or incomplete Cascade complex. (C) Activity assays with purified Cascade using in vitro transcribed α-32P–uridine triphosphate–labeled pre-crRNA from E. coli K12 (repeat sequence: GAGUUCCCCGCCAGCGGGGAUAAACCG), E. coli UTI89 (repeat sequence: GUUCACUGCCGUACAGGCAGCUUAGAAA), and non-crRNA as substrates. (D) Activity assays as shown in (C) for 15 min with purified MalE-LacZα and MalE-CasE fusion proteins. (E) Northern blot as shown in (B) with Cascade or Cascade-CasEH20A. (F) Activity assays as shown in (C) for 30 min with purified Cascade or Cascade-CasEH20A.