Verification of CRISPRi

To test CRISPRi, we have constructed a constitutive dCas9 on pRSFDuet, a constitutive sgRNA targeting mRFP (gR4mRFP) on pCDFDuet and a constitutive mRFP on pETDuet. Then we set up a strictly controlled experiment as follows:

  • Case: constitutive dCas9 on pRSFDuet + a constitutive gR4mRFP on pCDFDuet + constitutive mRFP on pETDuet
  • Controll: constitutive dCas9 on pRSFDuet + an empty pCDFDuet + constitutive mRFP on pETDuet

CRISPRi test.png
As expected, mRFP repressed by CRISPRi (dCas9 and gR4mRFP) is not as red as the control.

The sequence of gR4mRPF is given in our part BBa_K1026003 . This sequence comes from the following literature: QI, LEI S., LARSON, MATTHEW H., GILBERT, LUKE A., DOUDNA, JENNIFER A., WEISSMAN, JONATHAN S., ARKIN, ADAM P. & LIM, WENDELL A. 2013. Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell, 152, 1173-1183.

And CAUTION that:

  • There shall not be extra nucleotides between promoter and sgRNA itself.

gRNA shall be exactly transcribed out. Any extra nucleotides would potentially jeopardize the normal function of the gRNA. Therefore, BioBrick scars are not allowed here. Instead of cut-and-ligation, inverse PCR or In-Fusion is preferred for the assembly task. Our part BBa_K1026003 can be an example of no-scar assembly.

  • A reliable terminator is applied here to ensure the termination of sgRNA.