To generate suitable gRNAs for cas12a assay, trnL region of the four species was conducted for multiple alignment by MultAlin (http://multalin.toulouse.inra.fr/multalin/ )32 (link). There were two significant points as guideline for gRNA design for species differentiation: (1) searching for protospacer adjacent motif (TTTV (V = A, G or C)) and (2) the sequences for DNA targets having variation among four species in the seed sequences (1–5 first bases next to PAM)25 (link), given as gRNA-A and gRNA-B for specific P. amarus (Fig. 1 A). The synthesis of gRNA was done by in vitro transcription (IVT) under double stranded (ds) DNA as a template. The dsDNA was constructed and synthesized from Integrated DNA technologies (IDT, USA) which consisted of three parts as (1) T7 promoter regions, (2) tracrRNA to incorporate with cas12a to form binary complex and (3) crRNA to bind with DNA target, forming a tertiary complex. These dsDNAs were used as template for RNA synthesis via in vitro transcription (IVT) by HiScribe T7 Quick (#E2050S, NEB, US). The synthetic gRNAs were purified to remove the impurities by the Monarch RNA Cleanup Kit (50 µg) (NEB, US). The synthetic gRNA products were measured for amount and purity by Nanodrop and 2% agarose gel electrophoresis and then adjusted for concentration to 10 µM for further study.
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