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 placed ssDNA, ssRNA, and phosphorothioate ssDNA extensions of various lengths ranging from 7 to 31 nucleotides on either the 3'-or 5'-ends of the crRNA targeting GFP (green fluorescent protein), referred to here as crGFP (Fig. 1b-h) . In order to measure the collateral or trans-cleavage activity of Cas12a, we employed a FRET-based reporter used in DETECTR, 1 composed of a fluorophore (FAM) and a quencher (3IABkFQ) connected by a 5-nucleotide sequence (TTATT), which displays increased fluorescence upon cleavage. Consistent with the previous literature 13 , respectively. The fold in fluorescence was normalized by taking the ratio of background-corrected fluorescence signals of sample with activator to the corresponding sample without activator. Error bars represent ± SEM, where n = 6 replicates; two-way ANOVA test two-way ANOVA (n=3, N=2, ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). The experiments were repeated at least twice with n = 3 per experiment. when using wild-type crRNAs, we observed that the LbCas12a exhibited higher trans-cleavage activity than the AsCas12a or the FnCas12a, and therefore, we designed various modified crRNAs compatible with LbCas12a. Using the same reporters, we discovered that ssDNA and ssRNA extensions on the 3'-end of crGFP markedly enhanced the trans-cleavage ability of target-activated LbCas12a. Comparing the two types, the ssDNA extensions demonstrated higher activity than the corresponding ssRNA (Figs. 1b-d,f and Supplementary Figs. [1] [2] [3] [4] [5] . On the other hand, the phosphorothioate ssDNA extensions at the 3'-or 5'-end displayed minimal or no activity, showing decreased fluorescence intensity as modification length increased (Figs. 1e,h and Supplementary Figs. [1] [2] [3] [4] [5] . This observation suggests that further extending the crRNA with 13-mer phosphorothioate ssDNA and beyond significantly inhibits LbCas12a trans-cleavage activity. The finding corroborated B. Li and colleagues that phosphorothioate ssDNA may prevent crRNA-Cas12a-DNA complex formation. 14 Notably, the 3'-DNA with 7-mer extensions on the crGFP, referred as crGFP+3'DNA7, yielded the highest fluorescence signal compared to other modifications, measuring approximately 3.5fold higher intensity than the wild-type crGFP (Fig. 1c, Supplementary Figs. 1a,2a) . By investigating the conformational changes from the crystal structure of the binary LbCas12a:crRNA complex 12, 15, 16 , we observed that the 3'-end modifications on crRNA is proximal to the RuvC region of the LbCas12a. This supports our observation that the 3'-end extensions lead to higher trans-cleavage activity than the 5'-end. We speculated that once an R-loop is formed between crRNA and dsDNA or ssDNA activator, the LbCas12a executes a partial trans-cleavage of the 3'end of crRNA, leaving an overhang. These remaining extensions may further expand the nuclease domain in the LbCas12a, resulting in conformational changes and allowing more access for nonspecific ssDNA cleavage. To confirm our hypothesis, we attached different fluorophores, or fluorophore-quencher pairs separated by DNA linkers, to either the 3'-or 5'-end of the crGFP with 7-mer DNA extensions and analyzed by denaturing gel electrophoresis. Surprisingly, we discovered that the 3'-end of the crRNA is processed by LbCas12a only in the presence of an activator while the 5'-end is cleaved by LbCas12a even in the absence of the activator (Fig. 2a,b and Supplementary Figs. 6,7).