Figure 3.

Abundance of >26-nt sRNAs in AGO1 edited mutants. A) Schematic diagram of the AGO1 gene showing the Cas9 target region in exon 1. Short arrows indicate primers used for PCR analysis. In the wild-type sequence (top sequence), the target region is highlighted in light blue. The Cas9 cleavage site is indicated by a black arrowhead and the PAM is shown in blue font. Homology-directed repair of the DNA double-strand break, using an electroporated ssODN as template, introduces 8-bp changes (underlined red font) into the genome, including an in-frame stop codon (bottom sequence). B) The AGO1 target region was amplified by PCR with primers F1/R2 (annealing outside the edited sequence) or mut-F1/R2 (with one primer annealing exclusively to the edited sequence). The panels show representative inverted contrast images of PCR products resolved by agarose gel electrophoresis. CC-124, wild-type strain; ago1-45 and ago1-59, edited mutants with a disrupted AGO1 gene in the CC-124 background. C) RNA gel blot analysis of sRNAs isolated from the indicated strains and detected with probes specific for the most abundant >26-nt sRNAs of Clusters 51304+ or 69832−. The same membranes were reprobed with the U6 small nuclear RNA sequence as a control for equivalent loading of the lanes. The asterisks indicate putative RNA precursors of the >26-nt sRNAs. Examined cells were cultured under various nutritional conditions: Control, nutrient-replete medium; N starvation, nitrogen deprivation for 24 h; and S starvation, sulfur deprivation for 24 h. D) Size distribution of genome-mapped total sRNAs from wild type (CC-124) or ago1 mutant (ago1-45) strains. Data from replicate sRNA libraries are shown as bars of the same color.