In all five cases, Cas9 programmed with these chimeric RNAs efficiently cleaved the plasmid at the correct target site (Fig. 5D and fig. S15D), indicating that rational design of chimeric RNAs is robust and could, in principle, enable targeting of any DNA sequence of interest
The design of single "chimeric RNAs" was presented as an add on to the core findings of this paper. Today, the design of single guide RNA (sgRNA) and dual guide RNA (dgRNA) for CRISPR-Cas system has become a subfield of bioengineering that has inspired new basic biochemistry questions about the function of endogenous CRISPR-Cas systems. This month, a paper was published that identifies a new Cas9 structural motif called the guide repeat clasp (GRC) motif which helps to coordinate R-loop sensing and enable dsDNA cleavage (Chilamkurthy et al., 2026 Nucleic Acids Research). The research was motivated by observed differences between endogenous dgRNA and engineered sgRNA editing efficiency and specificity. It is really interesting to me that the authors pursued the design of single guide RNAs in this core paper and didn't save it for future research. It is one of the things that makes this paper excellent.