Towards Understanding Trans-Cleavage of Natural and Synthetic Nucleic Acids by Cas12a for Sensitive CRISPR Biosensing

CRISPR/Cas systems have been widely utilized for the development of biosensing platforms for precision molecular diagnostics. Their remarkable biosensing performance critically depends on the efficiency of sequence-independent trans-cleavage in type V and VI Cas effectors. Cas12a, a typical example of type V Cas effector exhibits varying trans-cleavage efficiency on different types of nucleic acids, and also in response to different nucleobase sequences. However, the underlying mechanism of Cas12a’s trans-cleavage characteristic remains unclear. To explore this mechanism, we introduced Xeno nucleic acids (XNA) as potential trans-cleavage substrates of Cas12a. XNAs are chemically modified nucleic acid analogues, which originate from chemical modifications of nucleobases, sugar moieties, and the backbone. We observed a progressive decrease in trans-cleavage rates by Cas12a across different types of XNAs, in the following sequence: nucleobase-modified XNA > sugar moiety-modified XNA > backbone-modified XNA. In addition, more complex chemical modifications on either of the three above locations led to the lowering of the trans-cleavage rate of Cas12a. These findings elucidate the mechanism behind Cas12a’ trans-cleavage characteristic, which is attributed to varying molecular complexity of the sugar moieties and nucleobases. Based on these findings, we also developed a colorimetric CRISPR/Cas12a biosensing system utilizing XNA for the detection of circulating tumor DNA (ctDNA), with a limit of detection of 10 pM and a 4 logs detection range from 10 pM to 100 nM. These results indicate that XNA can serve as a novel Cas12a trans-cleavage target for sensitive biosensing applications.