The iGEM Guelph 2021 team chose to improve two parts. The first was the CRISPR activation (CRISPRa) system designed to target the promoter region of CLE18. Five single guide RNAs were initially tested in the CRISPRa backbone, represented by the following parts:
The second was the CRISPRa system designed to target the promoter region of ICE2. Again, five single guide RNAs were initially tested in the CRISPRa backbone, represented by the following parts:
sgRNAs function by attaching to Cas9 proteins, and then guiding the Cas9 protein to a specific region of the DNA where the sgRNA binds to a complementary piece of DNA. This allows the Cas9 protein to perform its action. This project utilized a form of the Cas9 protein that lacked endonuclease activity (dCas9), with a transactivation domain attached (VP64 domain).
When the sgRNA BBa_K4058018/BBa_K4058019/BBa_K4058020/BBa_K4058021/BBa_K4058022 (CLE18) or BBa_K4058023/BBa_K4058024/BBa_K4058025/BBa_K4058026/BBa_K4058027 (ICE2) binds to the dCas9 protein with the VP64 domain forming a complex, it binds to a specific region in the promoter of the CLE18 or ICE2 gene. This causes the bound dCas9 to recruit RNA polymerase for increased transcription of the genes of interest.
However, the efficiency of upregulation observed in target genes through the use of a single sgRNA can be increased through the use of multiple sgRNAs targeted towards the promoter region of that same gene. This technique is termed multiplexing. This multiplex construct should allow for the dCas9 protein to bind more efficiently to the promoter, causing the overall increase in up regulation efficiency.
The Guelph iGEM team created a plasmid construct that incorporated all 5 of the sgRNAs targeted to the promoter of each gene of interest, to observe a greater amount of promoter activity. This increased activity should result in greater mRNA expression. The improved part for the CLE18 CRISPRa system is represented by part BBa_K4058014 and the improved part for the ICE2 system is represented by part BBa_K4058015.