CRISPR/Cas9 system for disruption of biochemical pathway for sterol synthesis in Artemisia annua L.

WHO recommends artemisinin-based combination therapy for curing malaria which is still a health problem in Indonesia. Artemisia annua L. is the primary source for artemisinin comes from subtropical China and India. Some introduction materials are grown in Indonesia, but they produce low concentration of artemisinin. Artemisinin synthesis uses the isoprenoid pathway, in which farnesyl diphospate (FDP) serves as the main precursor. FDP is the main precursor for sterol synthesis as well. The gene controls sterol biosynthesis is Squalene synthase ( SQS ). So then SQS is considered a competitive gene for artemisinin biosynthesis. CRISPR/cas9 is the most advanced system for genome editing, and can be used to induce a targeted-mutation. The aim of this research was to elevate Artemisinin content through disruption of the biochemical pathway of sterol synthesis using CRISPR/Cas9. Materials and methods used were as follows. Artemisia CRISPR/Cas9 seeds were processed and grown in selection medium and then soil. Seventy-seven Artemisia kanamycin resistant lines were produced. Fifty-two (of 77) lines were confirmed to have T-DNA by PCR with SQS-Fn and SQS-Rn primers, produced about 710 bp DNA fragment. By using SQS-Fs and SQS-Rs primers, those produced shorter DNA fragments (about 470 bp). A high-resolution electrophoresis QIAxcel was applied to identify for any nucleotide difference occured in PCR product of lines. Results showed that 44 lines (84.61%) have varied sizes with one to three nucleotides differences compared to control (plasmid, 468 bp). By assuming this analysis was right, it may be said that mutations has occured in SQS gene of Artemisia lines due to the CRISPR/Cas9.