Journal: eLife

Article Title: Molecular mechanism of active Cas7-11 in processing CRISPR RNA and interfering target RNA

doi: 10.7554/eLife.81678

Figure Lengend Snippet: ( a ) The mode of Cas7-like domain 1 (C7L.1) and C7L.2 interaction with the processed crRNA nucleotides –15 to –1 in both cartoon (top) and surface (bottom) representations. Key secondary elements involved in crRNA interaction are labeled. Insets indicate close-up views around U(–15)-U(–13)-G(–13), the C(–8)-A(–7)-C(–6)-G(–5) tight RNA turn, and the conserved A(–12)-U(–11)-G(–10)-U(–9) tetranucleotide. The catalytic residue His43 for crRNA processing is colored red. Dash lines indicate close polar contacts. ( b–c ) Top, various pre-crRNA used in processing reactions. Cyan colored triangles and dash lines indicate the pre-crRNA processing sites. Yellow bars indicate the sites of deoxy modification. The control RNA contains the last 14 nucleotides of the repeat plus the spacer. Spacer and repeat are shown in black and pink, respectively. ‘Csb pre-crRNA’ denotes the pre-crRNA for Candidatus Scalindua broadae Cas7-11. Processed products (P) of pre-crRNA are stained by SYBR Gold and imaged by ChemiDoc MP. Bottom, RNA processing results analyzed on polyacrylamide urea gel for the wild-type (1) and other pre-crRNA (2-8) by the wild-type Desulfonema ishimotonii Cas7-11 (DiCas7-11) (WT), the His43 to alanine mutant (H43A) of DiCas7-11, and the free-standing C7L.1 (fC7L.1). Processing products are indicated by cyan triangles. ( d ) Target RNA cleavage results analyzed on polyacrylamide urea gel using the wild-type and truncated pre-crRNA in the presence and absence of ethylenediaminetetraacetic acid (EDTA). ‘Cy3’ denotes the target RNA containing a 5’-Cy3 fluorophore. The cleavage products (P) of the Cy3-labeled target RNA are visualized on ChemiDoc MP using 550 nm as the excitation and 564 nm as the emission wavelength, respectively, and are indicated by green triangles. Figure 2—source data 1. Polyacrylamide gel image for deoxy precursor crRNA (pre-crRNA) processing activity shown in . Figure 2—source data 2. Polyacrylamide gel image for precursor crRNA (pre-crRNA) variant processing by DiCas7-11 and free-standing C7L.1 (fC7L.1) shown in . Figure 2—source data 3. Polyacrylamide gel image of target RNA cleavage activities with truncated crRNA and DiCas7-11 shown in .

Article Snippet: The gels were stained with SYBR Gold II (Invitrogen) stain and scanned by Bio-Rad ChemiDoc MP scanner.

Techniques: Labeling, Residue, Modification, Control, Staining, Mutagenesis, Activity Assay, Variant Assay

Journal: eLife

Article Title: Molecular mechanism of active Cas7-11 in processing CRISPR RNA and interfering target RNA

doi: 10.7554/eLife.81678

Figure Lengend Snippet: ( a ) Recognition of target RNA by the crRNA and Desulfonema ishimotonii Cas7-11 (DiCas7-11). The ferredoxin fold α1 and the thumb hairpin for each of the four Cas7-like (C7L) domains are shown as cartoons and colored as in . The quoted ‘thumb’ indicates the degenerate thumb motif for the C7L.4 domain. Insets show the two target cleavage sites in close-up views. RNA nucleotides and key amino acids are shown in stick models. The three atoms involved in formation of the ‘in-line’ geometry during phosphodiester bond breakage are labeled and indicated by thick dash lines. The closest of the three atoms to the putative catalytic residues, Asp654 (for site 2) and Asp429 (for site 1), are indicated by a connecting dash line. The close contact between Tyr360 and A(+4*) 2’-hydroxyl oxygen at site 1 and that between Lys754 and A(+9*) 2’-hydroxyl at site 2 are also indicated by dash lines. ( b ) Schematic of the Cy3-labeled (substrate 1) and other target RNA (substrates 2–7) used in cleavage activity assays. Yellow bars mark the locations of the deoxy modification on the target RNA substrate 3. Green colored triangles and dash lines indicate target RNA cleavage sites. ( c–f ) Target RNA cleavage by DiCas7-11 and its Tyr360 to alanine mutant (Y360A) are analyzed on polyacrylamide urea gel. The cleavage products (P) of the Cy3-labeled target RNA are visualized on ChemiDoc MP using 550 nm as the excitation and 564 nm as the emission wavelength, respectively. Cleavage products (P) of non-Cy3-labeled target RNA are stained by SYBR Gold and imaged by ChemiDoc MP. All cleavage products are indicated by green triangles. Figure 3—source data 1. Polyacrylamide gel image of target RNA cleavage activity by DiCas7-11 in presence and absence of metal ions shown in . Figure 3—source data 2. Polyacrylamide gel image of deoxy-target RNA cleavage activity by DiCas7-11 shown in . Figure 3—source data 3. Polyacrylamide gel image of target RNA variant cleavage activity by DiCas7-11 shown in . Figure 3—source data 4. Polyacrylamide gel image of target RNA cleavage activity by Desulfonema ishimotonii Cas7-11 (DiCas7-11) Y360A mutant shown in .

Article Snippet: The gels were stained with SYBR Gold II (Invitrogen) stain and scanned by Bio-Rad ChemiDoc MP scanner.

Techniques: Labeling, Activity Assay, Modification, Mutagenesis, Staining, Variant Assay

Journal: eLife

Article Title: Molecular mechanism of active Cas7-11 in processing CRISPR RNA and interfering target RNA

doi: 10.7554/eLife.81678

Figure Lengend Snippet: ( a ) Schematic of domain organization of wild-type and an insertion deletion variant DiCas7-11-Δint1. The region removed is colored in purple and numbered. ( b ) Cartoon representation of DiCas7-11 overlaying with density map resulted from focused classification using a mask around the insertion domain. The insertion structure model is from AlphaFold prediction. ( c ) Target RNA cleavage by DiCas7-11 (WT) and DiCas7-11-Δint1(Δint1) are analyzed on a polyacrylamide urea gel. Cleavage products (P) are stained by SYBR Gold and imaged by ChemiDoc MP and are indicated by green triangles. Figure 4—source data 1. Polyacrylamide gel image showing target RNA cleavage activity by DiCas7-11-Δint1.

Article Snippet: The gels were stained with SYBR Gold II (Invitrogen) stain and scanned by Bio-Rad ChemiDoc MP scanner.

Techniques: Variant Assay, Staining, Activity Assay