Trans-Nuclease Activity of Cas9 Activated by DNA or RNA Target Binding
In this study, we demonstrated that another type of Type II CRISPR-Cas system—the II type Cas9 system—also possesses “trans-cleavage” activity. This activity is guided by crRNA and tracrRNA and is activated via the binding of DNA or RNA targets. By combining the trans-cleavage activity of the Cas9 effector nuclease with nucleic acid amplification techniques, we created specific DNA and RNA sample detection platforms named DACD and RACD.
Cas9, under the guidance of chimeric sgRNA and nonchimeric crRNA with tracrRNA, exhibits significantly different ssDNA trans-cleavage activity. Enzyme kinetics analysis showed that the catalytic efficiency of Cas9 trans-cleavage guided by crRNA and tracrRNA is much higher than that of Cas9 trans-cleavage guided by chimeric sgRNA. This difference may be related to the different structural compositions of the SpyCas9-sgRNA-target DNA and SpyCas9-crRNA-tracrRNA-target DNA complexes. We observed that the SpyCas9-crRNA-tracrRNA-target DNA complex has a more homogeneous structural assembly compared to the SpyCas9-sgRNA-target DNA complex. Additionally, we noticed significant differences in the HNH domain between the two complexes, although the conformations of other domains are almost identical.
Similar to Cas12a, Cas9 also shows substrate sequence preference for trans-substrate cleavage. Both Cas9 and Cas12a nucleases preferentially degrade ssDNA substrates rich in T or C, suggesting that this might be a shared property of Type II and Type V CRISPR effector proteins. We verified the substrate sequence preference for Cas9-mediated trans-cleavage and cis-cleavage. While the HNH domain-dependent cis-cleavage might not have a sequence preference for the complementary strand, the RuvC domain-dependent cis-cleavage does show a sequence preference for the non-complementary strand, consistent with the non-specific ssDNA trans-degradation. These results suggest that the RuvC domain-dependent cis-cleavage and trans-cleavage might share a similar cleavage mechanism.
A detailed comparison of the trans-cleavage activities of Cas9, Cas12a, and Cas13a shows that although they exhibit different substrate binding affinities and different substrate catalytic efficiencies, they have similar detection limits in the limit of detection for amplified and free detection of DNA and RNA target oligonucleotides. Compared with platforms based on Cas12 and Cas13a, the Cas9-based detection platform may have some advantages.
Overall, our study provides new insights into Cas9-mediated nucleic acid detection, and also offers new considerations for the application of Cas9 in gene editing. Although our study only confirmed that Cas9 has trans-cleavage activity in vitro, we cannot rule out the possibility that this activity might also occur in vivo, potentially affecting gene editing. Therefore, further research is needed to determine whether this activity impacts the gene editing of other species when using CRISPR-Cas9.