Part:BBa_K3044016

Conjugative sgRNA-dCas9 system

Delivery of a CRISPR/dCas9 system via conjugation

(Designed and characterized by SDU-Denmark 2019)

SDU 2019 has assembled a sgRNA-dCas9 system with an oriT-R part (BBa_J01003) in order to make the sgRNA-dCas9 system transferrable via conjugation. However, the presence of a compatible conjugative plasmid is required for the efficient delivery. SDU 2019 used the RP4-8 plasmid for that purpose.

The dCas9 sequence is derived from part BBa_K1150000 codon optimized for E. coli. The dCas9 protein forms a complex with the sgRNA, which will then be able to downregulate the gfp gene (BBa_E0040).

dCas9 is an inactive form of the endonuclease Cas9. This means that the dCas9 does not make a double-stranded break in the target DNA as the Cas9 enzyme has the potential to do [1]. Instead, dCas9 sterically blocks the elongation of the transcription process when the sgRNA basepairs with the target DNA strand. Since the dCas9 enzyme does not make a break in the DNA, the gene will be left undestroyed. [2]

SDU 2019 demonstrated that the sgRNA-dCas9 system can be made transferrable via conjugation by the assembly of the oriT-R part BBa_J01003 into the sgRNA-dCas9 part BBa_K3044013 creating the transferrable sgRNA-dCas9 part BBa_K3044016. An RP4-8 plasmid was conjugated into the bacteria containing the part in order to be able to co-transfer the sgRNA-dCas9 plasmid into recipient cells. For the conjugation experiment described next, the donor strain contained the part BBa_K3044016 and the recipient strain contained the part BBa_K3044029.

Experimental procedure for the conjugation experiment
The donor and recipient bacteria were grown to late exponential phase and mixed in a 1:1 ratio. The conjugating samples were incubated at 37°C and plated on agar plates containing appropriate antibiotics, selecting for transconjugants containing the RP4-8 plasmid, the transferrable sgRNA-dCas9 system and the plasmid containing the gfp gene.

To verify the presence of the transferrable sgRNA-dCas9 plasmid in recipient bacteria already containing the plasmid that holds the gfp gene, PCR was performed by using primers that were designed to specifically hybridizing to both plasmids. Due to the large size of the dCas9 protein, a forward primer, designed to hybridize around position 2500 in the nucleotide sequence of the dCas9 gene in order to be able to distinguish the presence of the oriT sequence located downstream of the dCas9 sequence. Furthermore, a primer hybridizing to the prefix- and suffix site of the plasmid backbone was used for the PCR reaction. The gel picture indicated in figure 1 reveal the successful conjugation of the transferrable sgRNA-dCas9 system into recipient bacteria.

Figure 1. PCR products loaded on a 1% agarose gel. Lane 1-4 depicts the presence of the transferrable sgRNA-dCas9 plasmid (BBa_K3044016) received from the donor bacterium and the plasmid containing the gfp gene (BBa_K3044029)). Lane 5-8 verifies the presence of a transferrable dCas9 plasmid, lacking the sgRNA, that is received from the donor bacterium acting as a control for the action of dCas9. Lane 9-12 display the presence of the plasmid containing the gfp in recipient cells. The L indicates the 10 kb DNA size marker.

As the bands clearly verify in figure 1, the transconjugants containing both the transferrable sgRNA-dCas9 system and the plasmid containing the gfp gene.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 478
  Illegal BglII site found at 1552
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 4547
  Illegal NgoMIV site found at 4557
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 4449