Design and in vitro test of sgRNA for the CRISPR/Cas9 plasmid construct of the SQS gene of Artemisia annua L.

Artemisinin is the main compound of the dihydroartemisinic acid (DHA)-based antimalarial drug. Its synthesis might be impeded by squalene synthase (SQS) gene. This study aimed to design and synthesis sgRNA of SQS gene of Artemisia annua L., to amplify DNA fragment of the target gene, and to set up in vitro test to finding out sgRNA function. The CRISPR/Cas9 system is applied as it provides a more precise modification of the gene sequences. This preliminary study started with designing single guide RNA (sgRNA) and investigating their functions in directing the Cas9 protein to cut the target. The methods included designing sgRNA and primers using available tools online, synthesizing sgRNA, amplifying SQS DNA target, and setting up in vitro testing using kits and according to manufacturer instructions. The experiment resulted in two sgRNAs and two SQS DNA fragments (targets) that were incubated with the Cas9 protein at 37°C for 3 h in the in vitro test reaction. Both sgRNAs functioned since both 1.6 and 1.4 kbp DNA fragments became two smaller DNA fragments. These indicate that the CRISPR/Cas9 vector construct to target the SQS gene in the A. annua plant can use these two sgRNAs.