Targeted genome editing using CRISPR/Cas9 has been on the forefront of genome editing research lately. The Cas9 system is effective, efficient, and reliably specific in its approach to cleavage of nucleotides of interest. The catalytic activity of Cas9 depends on its ability to recognize short DNA sequences called the protospacer adjacent motif (PAM). Although it’s already emerging as a promising tool, Cas9 genome editing approaches have been largely limited to DNA substrates.
Recent evidence suggested that single-stranded DNA (ssDNA) Cas9 substrates can be activated for cleavage by a separate PAM-presenting oligonucleotides (PAMmers). Additionally, the same domains that act on these ssDNA substrates, have been shown to catalytically act on RNA in a similar fashion. This insightful evidence triggered O’Connell and colleagues at the University of California Berkeley to explore the nuclease activity of the Cas9 system on single-stranded RNA (ssRNA).
Equipped with a curious zest, O’Connell and colleagues at UC Berkeley set out to decipher the possibilities of Cas9-mediated cleavage of single-stranded RNAs (ssRNAs) by conducting in vitro Cas9-mediated dual-guide RNA cleavage experiments using both RNA and DNA substrates.
Additionally, the team wanted to understand sequence specific interaction between Cas9 and RNA, the binding affinity of the PAMmer-ssRNA complex, and factors that affect PAMmer nuclease activation.
Here’s what this adventure revealed:
- PAMmers consisting of deoxyribonucleotides activated Cas9 for the specific cleavage of ssRNA
- RNA cleavage did not occur when ribonucleotide derived PAMmers were used, insinuating Cas9 recognizes the helical structure or deoxyribonucleotide moieties within PAM
- dsDNA substrates could not be cleaved and RNA-DNA hybrids were only cleaved when the non-target strand was composed solely of deoxyribonucleotides
- Cas9 could not cleave ssRNA at temperatures lower than 37 and the abundant presence of di- or tri-deoxyribonucleotides in solution led to poor activation of the Cas9 cleavage system
- PAMmer-mediated nuclease activation of Cas9 was not affected by chemical modifications to the PAMmer.
- Cas9-guide RNA (gRNA) complex cleaves PAMmer-ssRNA targets regardless of PAM basepairing
Moreover, the team surmised that these findings can be used to purify endogenous untagged RNA transcripts under physiological conditions without crosslinking requirements or the need for large sets of biotinylated probes.
Feel free to ingest and digest more of this exciting brain nourishment at Nature 2014.