Cas10-Csm Complex crRNA Profiling

Total crRNAs were extracted from purified Cas10-Csm complexes as described previously in Chou-Zheng and Hatoum-Aslan, 2019 (link) with slight modifications. Briefly, 300–600 pmols of purified complexes were resuspended in 750 µl TRIzol Reagent (Invitrogen, NY) and subsequent RNA extraction steps were completed as recommended by the manufacturer. Extracted crRNAs were end-labeled with T4 Polynucleotide Kinase in a reaction containing γ-[³²P]-ATP (PerkinElmer, MA), and resolved on an 8% Urea PAGE. The gel was exposed to a storage phosphor screen and visualized using an Amersham Typhoon biomolecular imager (Cytiva, MA). For densitometric analysis, the ImageQuant software was used. Percent of intermediate crRNAs was obtained using the following equation: [intensity of intermediate crRNA signal (71 nt) ÷ sum of signal intensities for the dominant crRNA species (71 nt +43 nt + 37 nt + 31 nt)]×100%. The data reported represents mean values (± SD) of 2–4 independent trials (see appropriate figure legends for details).