Fig. 5: Cas6-2 can assemble with the Cas subunits of the type I-B system into an inactive Cascade complex. | Communications Biology

Fig. 5: Cas6-2 can assemble with the Cas subunits of the type I-B system into an inactive Cascade complex.

From: A type III-associated Cas6 functions as a negative regulator of type I-B CRISPR-Cas system in Thermus thermophilus

Fig. 5

a Strategy for copurification of native I-B Cascade complex using pRKP31-PEDVSp3-Cas8His plasmid. b The SEC chromatogram of the Cascade complex purified by a C-terminal His-tagged Cas8b in the wild-type strain (Cascade-WT). c The SEC chromatogram of the Cascade complex purified by a C-terminal His-tagged Cas8b in the HB27Δcas6-1 strain (Cascade-Δ6-1). d The Coomassie-stained SDS-PAGE gel of Cascade complex samples, obtained by concentrating the elution peaks in (b, c). The subunits of the two Cascade complexes are indicated with arrows. e Histogram of the distribution of crRNAs present in I-B Cascade complex over the 11 CRISPR arrays and the artificial mini-CRISPR array. f Schematic of in vitro dsDNA cleavage assay of type I-B Cascade complex. The 5’ FAM labeled non-target strand or target strand is shown. g, h Cleavage of dsDNA mediated by Cascade-WT complex in the absence or presence of ATP. i, j Cleavage of dsDNA mediated by Cascade-Δ6-1 complex in the absence or presence of ATP. The 5’ FAM-labeled non-target strand or target strand, is indicated at the bottom of each panel. Time duration (minutes) is shown.

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