Journal: bioRxiv

Article Title: Activation and self-inactivation mechanisms of the cyclic oligoadenylate-dependent CRISPR ribonuclease Csm6

doi: 10.1101/2020.02.19.952960

Figure Lengend Snippet: ( a, b ) Overall structure of EiCsm6 homodimer bound to cFA6, shown in two orientations (a: side view of the EiCsm6 homodimer; b: view of the cFA6 binding site at the CARF domain interface). The EiCsm6 homodimer is shown in cartoon representation (with protomers colored light and dark blue). cFA6 is depicted in sphere format. ( c ) Zoom-in view of the cA6 binding site at the CARF domain interface of EiCsm6, showing a 2m F O -D F C composite omit map, contoured at 1.0 σ and displayed within a radius of 2.2 Å around cFA6.

Article Snippet: The gene sequence of EiCsm6 was amplified from genomic DNA ( Enterococcus italicus DSM-15952) by PCR and cloned into a 1B vector (Addgene 29653) using ligation-independent cloning resulting in constructs carrying an N-terminal hexahistidine tag followed by a Tobacco Etch Virus (TEV) protease cleavage site.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Activation and self-inactivation mechanisms of the cyclic oligoadenylate-dependent CRISPR ribonuclease Csm6

doi: 10.1101/2020.02.19.952960

Figure Lengend Snippet: ( a, b, c ) Protein-cFA6 interactions. EiCsm6 is depicted in cartoon representation; cFA6 is shown as sticks. Hydrogen bonding interactions are represented as black dotted lines. ( d ) Fluorogenic RNase activity assay of CARF domain mutants of EiCsm6 (0.5 nM) in the presence of 10 nM cyclic-hexa-AMP (cA6). AU, arbitrary units. ( e ) Growth curves (optical density at a wavelength of 600 nm) of Staphylococcus aureus strains harboring pTarget and pCRISPR plasmids. The pCRISPR plasmid expresses wild-type or CARF domain mutants of EiCsm6 and the Staphylococcus epidermidis Cas10-Csm effector complex containing an inactivating point mutation in the HD domain of Cas10. Data points in panels d and e represent the mean of three replicates; error bars represent the standard error of the mean (s.e.m.). Source data are provided as a Source Data file.

Article Snippet: The gene sequence of EiCsm6 was amplified from genomic DNA ( Enterococcus italicus DSM-15952) by PCR and cloned into a 1B vector (Addgene 29653) using ligation-independent cloning resulting in constructs carrying an N-terminal hexahistidine tag followed by a Tobacco Etch Virus (TEV) protease cleavage site.

Techniques: Activity Assay, Plasmid Preparation, Mutagenesis

Journal: bioRxiv

Article Title: Activation and self-inactivation mechanisms of the cyclic oligoadenylate-dependent CRISPR ribonuclease Csm6

doi: 10.1101/2020.02.19.952960

Figure Lengend Snippet: ( a ) Zoom-in view of nucleotides A1 and A6 of cFA6 bound to the EiCsm6 CARF domain. Residues implicated in the cleavage are represented as sticks; hydrogen bonding interactions are depicted as black dotted lines. The red arrows represent the proposed reaction mechanism of cA6 cleavage involving nucleophilic attack of the 2’-hydroxyl group of A6 onto the phosphodiester group connecting A6 and A1. ( b ) RNase activity assay comparing the activity of WT and T11A EiCsm6 proteins in the presence of 500 nM cA6. The black arrows represent spike-ins of 0.8 pmol of RNaseAlert substrate at 5 min intervals. AU, arbitrary units. ( c ) Growth curves (optical density at a wavelength of 600 nm) of Staphylococcus aureus strains harboring pTarget and pCRISPR plasmids. The pCRISPR plasmid expresses wild-type or CARF domain mutants of EiCsm6 and the Staphylococcus epidermidis Cas10-Csm effector complex containing an inactivating point mutation in the Csm3 subunit (D32A; dCsm3). Data points in panels b and c represent the mean of three replicates; error bars represent the standard error of the mean (s.e.m.). Source data are provided as a Source Data file.

Article Snippet: The gene sequence of EiCsm6 was amplified from genomic DNA ( Enterococcus italicus DSM-15952) by PCR and cloned into a 1B vector (Addgene 29653) using ligation-independent cloning resulting in constructs carrying an N-terminal hexahistidine tag followed by a Tobacco Etch Virus (TEV) protease cleavage site.

Techniques: Activity Assay, Plasmid Preparation, Mutagenesis

Journal: bioRxiv

Article Title: Activation and self-inactivation mechanisms of the cyclic oligoadenylate-dependent CRISPR ribonuclease Csm6

doi: 10.1101/2020.02.19.952960

Figure Lengend Snippet: ( a ) Zoom-in view of the HEPN domain ribonuclease active site in EiCsm6. The conserved R372, N373, H377 residues of the HEPN catalytic motif, and additionally R274, are represented as sticks. Electron density, interpreted as a sulfate ion (depicted as 2m F O -D F C composite omit map, contoured at 1.0 σ and displayed within a radius of 2.2 Å), is located within hydrogen bonding distance of H377, likely mimicking the scissile phosphate group of an RNA substrate. ( b ) Schematic model of the cA6-mediated allosteric activation mechanism of Csm6 enzymes.

Article Snippet: The gene sequence of EiCsm6 was amplified from genomic DNA ( Enterococcus italicus DSM-15952) by PCR and cloned into a 1B vector (Addgene 29653) using ligation-independent cloning resulting in constructs carrying an N-terminal hexahistidine tag followed by a Tobacco Etch Virus (TEV) protease cleavage site.

Techniques: Activation Assay