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Development of split Cas6 for inducible dimerization modules
Author(s) -
Ling Justin A.,
Vontalge Emma J.,
Sashital Dipali G.
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.649.10
Subject(s) - crispr , rna , förster resonance energy transfer , thermus thermophilus , guide rna , biology , ctd , palindrome , endoribonuclease , rna binding protein , computational biology , genetics , cas9 , microbiology and biotechnology , chemistry , gene , fluorescence , physics , rnase p , oceanography , escherichia coli , quantum mechanics , geology
Cas6 endoribonucleases are essential for guide RNA biogenesis during CRISPR‐Cas (clustered regularly interspaced short palindromic repeats‐CRISPR associated) immunity, and have exquisite specificity for short structured RNAs that form in the repeating regions of CRISPR transcripts. The Cas6 protein from Thermus thermophilus contains two domains, an N‐terminal domain (NTD) containing the endoribonuclease active site, and a C‐terminal RNA‐binding domain (CTD). The modular nature of this protein makes it a potential platform for molecular scaffolding by splitting the two domains and inducing dimerization through RNA binding. Here we show that the Cas6 NTD and CTD can be split into two separate proteins, and that the domains retain their RNA binding and cleavage activity. Fusion of the domains to the C‐termini of cyan or yellow fluorescent protein (CFP or YFP) enabled measurement of dimerization in the absence or presence of RNA using fluorescence resonance energy transfer (FRET). We find that wild‐type Cas6 NTD and CTD dimerize even in the absence of RNA. However, introduction of mutations in the interface between the two domains disrupts this interaction. The interface mutations do not disrupt RNA binding or cleavage, and addition of RNA restores dimerization of the two domains. These results demonstrate that split Cas6 can be developed into a molecular scaffolding platform that can be controlled through the addition of RNA. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .