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. 2016 May 23;44(12):5872–5882. doi: 10.1093/nar/gkw469

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© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 4. — Purification of recombinant synthetic Cascade variants with shortened and elongated spacers. Variants of crRNAs with wild type (WT) spacer (32 nt), short spacer length (14 nt) and long spacer length (50 nt) were designed. Production and maturation of the crRNA variants in E. coli was verified. Recombinant Cascade complexes were produced and purified via size-exclusion chromatography. Cas7fv filaments (peak 1) and Cas5fv-Cas7fv dimers (peak 3) were observed and the middle peak corresponded to fully assembled Cascade ribonucleoproteins (peak 2). The relative shift of this peak during identical size-exclusion chromatography runs and SDS-PAGE revealed that additional spacer nucleotides result in additional Cas7fv subunits.