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.

This is a really elegant experiment; editing the reporter gene (GFP) is an easy way to verify the activity. It can also be done in vitro which eliminates a lot of possible confounding variables in the experiment. Eliminating confounders can enable drawing conclusions with less experiments, in vivo experiments can often require secondary and tertiary evidence to please the reviewers. Other authors have used similar approaches to verify TF-promoter interactions using plasmids transformed into a Nicotiana benthamiana system (Chenxin et al., 2026 New Phytologist). This experiment used reporters to verify TF-promoter interactions from Catharanthus roseus outside of their native environment. Although the experiments were different, both are creative uses of reporters and synthetic biology (plasmids) to confirm biochemical interactions and functions of particular molecules.

"Possibly strand separation" The authors do a good job here of not over-claiming what the results from these experiments are unable show. These results in aggregate show which sequences are needed for DNA cleavage and suggest a role in specifically dsDNA editing, but do not show the mechanism for opening the dsDNA strands.

This helps us to understand that the PAM has a very specific function in the CRISPR-Cas system. It enables the selective editing of dsDNA, specifically the DNA in foreign sequences (self sequences won't have a PAM next to the target). ssDNA is not as common so the system being able to edit ssRNA but not dsDNA without a PAM sequence is not surprising because there is no clear mechanism for evolutionary feedback to evolve a mechanism to prevent self targeting of ssDNA.