Single-stranded RNA (ssRNA) (ssRNA)

Single-stranded RNA (ssRNA) substrates, when targeted in the presence of a complementary guide RNA and a PAM-presenting DNA oligonucleotide (PAMmer), represent a specialized therapeutic target for the CRISPR-Cas9 system. While the Streptococcus pyogenes Cas9 (SpCas9) protein naturally targets double-stranded DNA, the use of a PAMmer—a short DNA oligonucleotide that provides a Protospacer Adjacent Motif (PAM) in trans—reprograms the enzyme to recognize and cleave ssRNA sequences (O'Connell et al., 2014, Nature). This mechanism allows for the programmable manipulation of specific RNA species without permanently altering the host genome. Biologically, these RNA targets include messenger RNA (mRNA), non-coding RNA, and viral RNA, which play critical roles in protein synthesis and cellular regulation. In disease contexts, this system has been explored to eliminate toxic RNA repeats in conditions like myotonic dystrophy and to target the genomes of RNA viruses (Batra et al., 2017, Cell). Therapeutic challenges include ensuring high specificity to avoid off-target RNA cleavage and the complex delivery requirements of the Cas9 protein, guide RNA, and PAMmer components (Nelles et al., 2016, Cell). This approach expands the CRISPR toolkit beyond genome editing to include precise transcriptome engineering.

Other names

Target RNARNA substrateMessenger RNANon-coding RNAViral RNATranscriptome target

Sequence-specific RNA binding and endonucleolytic cleavage mediated by the Cas9-sgRNA-PAMmer complex.