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

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

We speculated that the reporter composition itself may affect the LbCas12a collateral cleavage activity. Therefore, we incorporated and tested various nucleotides (GC and TA-rich) and fluorophores (FAM, HEX, and Cy5) within the reporter. Consistent with our hypothesis, we observed that the LbCas12a achieved maximal trans-cleavage activity with FAM or HEX and TArich reporter ( Fig. 2f and Supplementary Figs. [1] [2] [3] [4] [5] . Furthermore, these results led us to question whether the trans-cleavage activity is dependent on the sequence of ssDNA extensions on 3'-end of the crRNA. To test this, we altered the nucleotide content of the extended regions of the crGFP. It turned out that the crGFP with TA-rich extensions carried out significantly more collateral cleavage than those with GC-rich regions ( Fig. 2h and Supplementary Fig. 8 ).