Part:BBa_K4058008
ICE2 sgRNA D for CRISPRa
This part is a single guide RNA (sgRNA) sequence designed to direct a CRISPR-dCas9 transcriptional activation system (CRISPRa system) to bind to the promoter region of the gene ICE2. The iGEM Guelph 2021 team designed this system to upregulate transcription from the gene ICE2, which is involved in the cold-stress response pathway in the model organism Arabidopsis thaliana.
Five target sequences were chosen from the promoter region of ICE2 in order to test for sgRNA binding efficiency. These sequences were termed ICE2 guide A, B, C, D, and E. This part contains guide D followed by a sgRNA scaffold. It is flanked on both ends by a BsaI type IIS restriction site plus adjacent bases that, when cleaved by BsaI, produce complementary overhangs to the CRISPRa plasmid pHSN6A01 when pHSN6A01 is also cleaved using BsaI. In this way, each sgRNA was inserted into pHSN6A01 via golden gate cloning. Each plasmid was then transformed into E. coli DH5 alpha and plated on LB media with kanamycin. The presence of the modified pHSN6A01 plasmid in E. coli colonies was confirmed via PCR. Each plasmid was subsequently transformed into Agrobacterium tumefaciens, plated on LB media with kanamycin and gentamicin, and the presence of the plasmid was once again confirmed via PCR. Next, Agrobacterium tumefaciens cells containing the plasmid were floral dipped into Arabidopsis thaliana plants. Seeds produced by the floral dipped plants were screened for the presence of the plasmid by plating on MS media with hygromycin. Screening revealed no positive transformants from the transformation using Agrobacterium tumefaciens containing ICE2 sgRNA D.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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