Towards understanding Trans-cleavage of natural and synthetic nucleic acids by Cas12a for sensitive CRISPR biosensing

• XNAs represent alternative trans -cleavage substrates for Cas12a. • Cas12a demonstrates varying XNA trans -cleavage rates in the following order: nucleobase modified XNA > sugar moieties modified XNA > backbone modified XNA. • More complex chemical modifications on nucleobases, sugar moieties or backbone lead to a more pronounced lowering of Cas12a trans -cleavage rates. • The biosensing performance of the CRISPR/Cas12a systems based on XNA is comparable to that of equivalent conventional DNA-based systems. 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 its higher affinity of Cas12a for substrates with simpler chemical structures, leading to increased trans -cleavage efficiency. 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 substrate for sensitive biosensing applications.