Neo hts

There are two components of Cas12a crRNAs: the universal scaffold region (UAAUUUCUA CUAAGUGUAGAU) for Cas12a protein recognition and binding and a customized region added to the 3′ end of the scaffold that provides specificity to the target sequences (the same sequence as the region following a TTTV PAM sequence). The design principle was consistent with the "CRISPR-SHERLOCK" method published by Zhang et al. 46 (link) The crRNA targeting the L108F mutation of the MPXV F8L gene was provided by Sangon Biotech (Shanghai) Co., Ltd. (see Table S1 for the crRNA sequence).
The specificity of crRNAs for different DNA templates was validated by using the CRISPR/Cas12a assay. In 10 μL volumes, 100 nM Cas12a (NEB), 100 nM crRNA, 1× NEB 2.1 buffer (NEB), 500 nM ssDNA fluorescent quenched reporter (5′ 6-FAM/TTATT/BHQ-1 3′, Sangon), and different concentrations of target dsDNA sequences diluted in RNase-free water were used. Reactions were incubated at 37 °C for 30 min, and real-time fluorescence was measured using a BioTek NEO HTS plate reader (BioTek Instruments, Winooski, VT, USA) with readings every 2 min (excitation: 485 nm; emission: 528 nm). The cis-cleavage of the sequences was further verified by electrophoresis on a 2% agarose gel at 120 V for 30 min (Figure S1).