Part:BBa_K3249006

MM CXCR4 sgRNA 3

Usage and Biology

This part consists of a 20 bp targeting sequence for a single guide RNA (sgRNA) designed to attach to the promoter region of the gene CXCR4 in the organism 𝘔𝘶𝘴 𝘮𝘶𝘴𝘤𝘶𝘭𝘶𝘴. When combined with a dCas9 protein this system can effectively alter transcription of the gene CXCR4.

Characterization

In order to determine whether or not our sgRNA is functional, we transiently cotransfected NIH 3T3 cells with 2 separate plasmids, one containing the MM CXCR4 sgRNA and the other containing a dCas9 protein linked to a VPR transcriptional activator domain. If both plasmids were successfully transfected into the cells then we expect to see an increase in CXCR4 transcription. Cells were incubated at 37° C for 48 hours to provide the cells enough time to adhere and express the gene. We then extracted the RNA from the NIH 3T3 cells and used reverse transcriptase to convert this RNA into cDNA. To verify that the sgRNA was recruiting the dCas9-VPR to the correct location, we performed qPCR to quantify the amount of CXCR4 mRNA present in each 24 well plate. Fold increase of CXCR4 expression shown below was evaluated using qPCR data.

Figure 1: Fold increase of CXCR4 transcription in NIH 3T3 cells when transiently cotransfected with dCas9-VPR and sgRNA 3.

Fold increase was calculated using the delta-delta Ct method for qPCR. All data was normalized to the reference genes recommended by MIQE guidelines and fold increase was compared to untransfected cells. A more in depth description of experimental conditions and data analysis can be found on the UC Davis 2019 iGEM wiki.

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]