Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection

Long T. Nguyen; Brianna M Smith; Piyush K. Jain

Structural analysis of LbCas12a shows that it contains a single RuvC domain, which processes precursor crRNA into mature crRNA, cleaves target dsDNA or ssDNA (referred here as activators), and executes nonspecific cleavage afterwards. 19, 20 Therefore, we were interested in understanding the effects of these modified crRNAs on cis-cleavage compared to the wild-type crRNA, as well as how cis-cleavage activity correlates to the trans-cleavage activity. Towards this, we carried out an in vitro cis-cleavage assay for various 3'-end and 5'-end modifications. We noticed that the cis-cleavage activity was either similar or marginally improved with most 3'-end modifications while the 5'-end modifications showed either similar or slightly reduced activity. This phenomenon suggests that the trans-cleavage activity is commensurate with the cis-cleavage activity .