Journal: Nature

Article Title: Two Distinct RNase Activities of CRISPR-C2c2 Enable Guide RNA Processing and RNA Detection

doi: 10.1038/nature19802

Figure Lengend Snippet: a, Illustration of LbuC2c2 RNA detection approach using a quenched fluorescent RNA reporter. b, Quantification of fluorescence signal generated by LbuC2c2 after 30 min for varying concentrations of target RNA in the presence of human total RNA. RNase A shown as positive RNA degradation control. (mean ± s.d., n = 3) c,. Quantification of fluorescence signal generated by LbuC2c2 loaded with a β-actin targeting crRNA after 3h for varying amounts of human total RNA or bacterial total RNA (as a β-actin null negative control). (mean ± s.d., n = 3) d, Tandem pre-crRNA processing also enables RNA detection. (mean ± s.d., n = 3) e, Model of the Type VI CRISPR pathway highlighting both of C2c2’s ribonuclease activities.

Article Snippet: The percent cleavage was determined as the ratio of total banding intensity for all shorter products relative to the uncleaved band and normalized for background within each measured substrate using ImageQuant TL Software (GE Healthcare) and fit to a one phase exponential association using Prism (GraphPad). crRNA filter-binding assays Filter binding assays was carried out in RNA processing buffer (20 mM HEPES pH 6.8, 50 mM KCl, 5 mM MgCl 2 , 10 μg/mL BSA, 10 μg/mL yeast tRNA, 0.01% Igepal CA-630 and 5% glycerol).

Techniques: RNA Detection, Fluorescence, Generated, Negative Control, CRISPR