Cas9, a component of the genome editing tool CRISPR–Cas9, is a DNA endonuclease. It is targeted by guide RNAs (gRNAs), and its activity depends on recognizing a short sequence known as the protospacer adjacent motif (PAM). O'Connell et al. show that Cas9 also targets single-stranded RNA (ssRNA) with high affinity, if PAM-presenting oligonucleotides (PAMmers) are provided. Cleavage experiments in vitro revealed that deoxyribonucleotide (but not ribonucleotide)-based PAMmers activated Cas9 to cleave ssRNA, and that this depended on the stability of the PAMmer–ssRNA duplex. Similar to DNA cleavage, ssRNA cleavage by Cas9–gRNAs was specific and programmable. Importantly, incubating dsDNA targets lacking a PAM with PAMmer–ssRNA substrates resulted in the selective cleavage of the ssRNA, demonstrating that when targeting RNA transcripts, genomic loci could be excluded. Tailored RNA recognition and cleavage have immense potential for RNA research and therapeutics.