Peer Review Report For: In silico and in vitro analysis of THCA synthase gene in Moroccan Cannabis Sativa, L [version 1; peer review: 2 not approved]

Background: Cannabis ( Cannabis sativa, L.) is an archaic, most infamous plant with unique therapeutic characteristics, and great economic interest. The identification of its THCAS synthesizing enzyme was a great leap forward in Cannabis investigation. However, basic molecular biology for THCAS gene expression remains largely unstudied. Methods: In this paper, we explored the ability of bioinformatics resources in the design of molecular cloning protocol and prediction of three-dimensional structure of recombinant THCAS in Moroccan Cannabis sativa variety. We further described conventional experiments performed in vitro . We used specific markers to isolate the nucleotide sequence of THCAS from the leaves of Cannabis, without including the native signal sequence. Results: As a result, we found high sequence similarity with THCAS and the mRNA precursor of the same gene as previously reported. In addition, we predicted the structure of the recombinant THCAS using the organic crystal structure prediction method, the amino acid sequence reference, and the artificial intelligence technology. Conclusions: This study paves the way to successfully express recombinant THCAS enzyme in hosts like Pichia pastori .