Bar-cas12a, a novel and rapid method for plant species authentication in case of Phyllanthus amarus Schumach. & Thonn.

Rapid and accurate species diagnosis accelerates performance in numerous biological fields and associated areas. However, morphology-based species taxonomy/identification might hinder study and lead to ambiguous results. DNA barcodes (Bar) has been employed extensively for plant species identification. Recently, CRISPR-cas system can be applied for diagnostic tool to detect pathogen's DNA based on the collateral activity of cas12a or cas13. Here, we developed barcode-coupled with cas12a assay, "Bar-cas12a" for species authentication using Phyllanthus amarus as a model. The gRNAs were designed from trnL region, namely gRNA-A and gRNA-B. As a result, gRNA-A was highly specific to P. amarus amplified by RPA in contrast to gRNA-B even in contaminated condition. Apart from the large variation of gRNA-A binding in DNA target, cas12a- specific PAM's gRNA-A as TTTN can be found only in P. amarus. PAM site may be recognized one of the potential regions for increasing specificity to authenticate species. In addition, the sensitivity of Bar-cas12a using both gRNAs gave the same detection limit at 0.8 fg and it was 1,000 times more sensitive compared to agarose gel electrophoresis. This approach displayed the accuracy degree of 90% for species authentication. Overall, Bar-cas12a using trnL-designed gRNA offer a highly specific, sensitive, speed, and simple approach for plant species authentication. Therefore, the current method serves as a promising tool for species determination which is likely to be implemented for onsite testing.