Part:BBa_K4058000

CLE18 sgRNA A 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 CLE18. The iGEM Guelph 2021 team designed this system to upregulate transcription from CLE18, which is a root growth factor that regulates root length and branching in the model organism Arabidopsis thaliana.

Five target sequences were chosen from the promoter region of CLE18 in order to test for sgRNA binding efficiency. These sequences were termed CLE18 guide A, B, C, D, and E. Each sgRNA was ordered as two complementary single stranded oligos that when combined into one tube, formed a single double stranded oligo with a 4bp overhang on either side. These 4bp overhangs were designed to be complementary to our BsaI-digested vector pHSN6A01, which allowed for Golden Gate cloning to be used to piece the sgRNA and the rest of the vector together. 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 CLE18 sgRNA A. However, two positive transformants were identified from the seeds transformed using Agrobacterium tumefaciens containing CLE18 sgRNA A.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]