Fig. 2

Reconstitution of DNA interference of the Aa-CRISPR-Cas system in vitro. a SDS-PAGE of purified Cas2/3 protein and WT Cascade complex preparations. Expected molecular weights for Cas2/3-His and Cascade-forming Cas8f1, Cas5f1, Cas7f1, and His-Cas6f proteins are approximately 127, 52, 36, 38, and 23 kDa, respectively. b crRNA of the type I-E S. thermophilus Cascade complex is shown for molecular weight comparison (lane I-E). Length of the respective crRNA is indicated below the gel. c ssDNA nuclease activity of Cas2/3. Cas2/3 nuclease activity assayed using M13mp18 (ssDNA) or pSP-CC (dsDNA) as substrates. d ATP hydrolysis by Cas2/3. Reaction rate constant k values (min⁻¹) of ATP hydrolysis by Cas2/3 were monitored in the presence of M13mp18 (ssDNA), pSP-CC (dsDNA), or in the absence of DNA (−). Error bars represent standard deviations for at least three separate experiments (individual data values are provided in Additional file 14). e DNA binding by Cascade. WT Cascade interaction with target DNA (SP-CC) bearing CC PAM and matching protospacer or non-target DNA containing defective AA PAM (SP-AA) or no matching sequence (NT) was assayed by EMSA in agarose gel. f Cascade-targeted dsDNA degradation by Cas2/3. Degradation of respective plasmids in the presence (+) or absence (−) of ATP and Cascade was initiated by addition of Cas2/3. g Cas2/3 cleavage within the R-loop. SP-CC oligoduplex ³²P-5′-labelled on either the non-target (NS) or target (TS) DNA strand was incubated with WT Cascade and Cas2/3 in the absence (−) or presence (+) of ATP. h Map of Cas2/3 cleavage sites. Arrows indicate cleavage positions within SP-CC sequence. The height of the arrow correlates with a relative amount of cleavage product (for more details see Additional file 5: Figure S4)