Cleavage efficiency of CRISPR/Cas9 system with G-quadruplex-capped single-guide RNA motifs in RNase II and RNase R

Modification of sgRNA has been considered as a necessary approach to enhance the stability and cleavage efficiency of the CRISPR/Cas9 system. In this study, a rigid G-quadruplex structure was genetically applied to the 3' end of typical sgRNA for protection of RNA from 3'-5' exoribonuclease degradation. The in vitro transcriptional production yields of sgRNAs bearing G-quadruplex structure such as sgRNA3 and sgRNA4 were around 1.4 and 1.5 times higher than the yield of typical sgRNA1, respectively. The results have also shown that appending G-quadruplex motif at the 3' end of typical sgRNAs did minorly affect the cleavage activity of CRISPR/Cas9. Interestingly, cleavage efficiency of CRISPR/Cas9 system with sgRNAs bearing the rigid G-quadruplex was fully retained in the presence of 3'-5' exoribonucleases such as RNase II or RNase R. In contrast, the cleavage activity of CRISPR/Cas9 system with the typical sgRNA1 was significantly decreased in the same condition. This protection of sgRNA through G-quadruplex structure-based modifications might provide a potential approach for improving cleavage efficiency of CRISPR/Cas9 system in the exoribonuclease environment.

The online version contains supplementary material available at 10.1007/s13205-025-04354-x.