Fig. 1

Purification and activity of Lactococcus lactis type III-A (Csm) CRISPR-Cas system (a) Genome organization of Type III-A CRISPR-Cas system in pKLM plasmid of Lactococcus lactis. The locus harbors tandem copies of 36 nt repeats (diamonds) and spacers (rectangles) of varying lengths. b Schematic representation of pACYC expression plasmid harboring L. lactis CRISPR-Cas genes Csm1-6, Cas6, and CRISPR RNA locus in a HDV ribozyme construct. c Schematic representation of crRNA and various crRNA-target RNA duplexes used in the study (crRNA: CRISPR RNA, CTR: cognate target RNA, NTR: non-cognate target RNA, PTR: protospacer target RNA, ATR: anti-sense target RNA, DTR: deoxy-substituted target RNA, NTD: non-target DNA). The blue dashed lines indicate target RNA cleavage sites. Deoxynucleotides are highlighted in yellow. d Silver stain profile of purified LlCsm RNP showing Csm1-5 proteins and crRNA. e In vitro RNA cleavage assay by WT LlCsm complex. The reactions were performed at 37 °C for 20 min and contained 100 nM LlCsm complex and 500 nM target RNA. f In vitro DNA cleavage assay by WT LlCsm complex. The reactions were carried out with 100 nM LlCsm complex, 200 nM target RNA on M13mp18 at 37 °C for 10–90 min as indicated in a cleavage buffer containing 33 mM Tris-acetate pH 7.6 (at 32 °C), 66 mM potassium acetate, and 10 mM MnCl₂. g Urea-polyacrylamide gel electrophoresis (Urea-PAGE) showing cOA synthesis by LlCsm complex. The reactions were carried out at 37 °C overnight with 100 nM LlCsm complex, 200 nM target RNA, 500 μM cold ATP spiked with [α-³²P]-ATP in a buffer containing 33 mM Tris-acetate pH 7.6 (at 32 °C), 66 mM potassium acetate, and 10 mM MgCl₂. h In vivo plasmid interference assays with the wild type LlCsm harboring a non-cognate crRNA or the wild type LlCsm harboring a cognate crRNA. i Dual fluorescence assay using DNA and RNA oligo reporters with 5′ Alexa Fluor 594 fluorophore/3′ Iowa Black RQ quencher and 5′ 6-FAM fluorophore/3′ Iowa Black FQ quencher, respectively. The Csm1-mediated DNase and the Csm6-mediated RNase are simultaneously detected at 570/630 nm excitation/emission (red) and 480/530 nm excitation/emission (green) wavelengths, respectively. Target RNA (CTR) was used as the stimulator at 500 nM, LlCsm at 200 nM, LlCsm6 at 1 nM, and metal ions at 10 mM. Curves are averages with error bars of three technical replicates subtracted by the signals from water-stimulated reactions.