Part:BBa_K2538009

Synthetic minimal adenovirus major late promoter (pMLPm)

The synthetic promoter minimal adenovirus major late promoter (pMLPm) contains three repetitions of the "a1" sequence, which is complementary to the gRNA sequence, enabling staphylococcus aureus gRNA-dCas9-VP64 complex to target the pMLPm synthetic promoter.[1] When the transcription factor VP64 (an engineered tetramer of the herpes simplex VP16 transcriptional activator domain) is fused to dCas9 enzyme it can be guided to a specific location in the genome, in this manner we can exploit it to promote downstream translation. [2]

Usage and Biology

The gRNA-dCas9-VP64 complex will target the pMLPm synthetic promoter and promote the expression of exogenous reverse caspase3 that will lead to apoptotic death of reactive astrocyte cells. mCherry reporter protein is also expressed under the control of the pMLPm promoter, it is fused to the exogenous caspase3 with Thoseaasigna virus 2A (T2A) peptide that is cleaved during translation.[3]

Characterization

This part was used and validated by BGU 2018 team in the following constructs:

1. pSynt-CMV

HEK293 cell-line was used in order to examine our engineered system, in which dCas9-VP64-gRNA complex can target and activate the pMLPm promoter and to induce apoptotic death. mCherry is also expressed under the regulation of pMLPm promoter and can be used as an indicator for the regulatory activity of pMLPm.

Co-transfection of HEK293-cells by pSynt-CMV and lenti-viral vector: CMV-dCas9-VP64. In this experiment both the gRNA and the dCas9-VP64 are expressed by cytomegalovirus promoter (CMV). The dCas9-VP64-gRNA complex could activate the pMLPm promoter, since mCherry expression was detected in FACS ARIA III (Fig 1), fluorescent microscope (Fig 2), and by laser scanning confocal microscope (Fig 3).

Figure 1: Histogram analysis of viable HEK293 cells. Number of cells with mCherry expression. Blue - cells without transfection. Red – 48 hours post co-transfection with lenti-viral-vector: CMV-dCas9-VP64 and pSynt-CMV, 23.1% of viable cells expressed mCherry.

Figure 2: Images of mCherry in HEK293 cells 24 hours post co-transfection with lenti:CMV-dCas9-VP64 and pSynt-CMV. A – bright field. B – Fluorescent microscope.

Figure 3: Laser scanning confocal microscope (Zeiss LSN 880) images of mCherry in HEK293 cells 48 hours post co-transfection with lenti viral vector:CMV-dCas9-VP64 and pSynt-CMV . A – excitation: 514nm emission: 570-630nm, B – bright field, C – merged.

mCherry expression in HEK293 cells was validated:

• In FACS ARIA we were able to see that although we had low transfection efficiency, 23.1% of cells expressed mCherry.

• In fluorescent and confocal microscope we were able to detect mCherry fluorophore in several cells.

• Although, we do not have a proof of apoptosis, we can see some background red-fluorescence in our images, cells that undergone apoptosis will disperse their expressed mCherry in the media, thus the fluorescence will not be as focused as seen with localized living cells using confocal microscopy, rather there will be a background of red-fluorescence, hence we may cautiously say that some of the cells that have expressed mCherry, have undergone apoptosis as well. This notion will require further validation.

In this experiment we saw that pMLPm can be activated in HEK293 cells by dCas9-VP64-gRNA complex, since we were able to validate mCherry expression in several methods.

In HEK293 cells we did it when both gRNA and dCas9-VP64 are regulated by the CMV promoter, our next step is to test pMLPm promoter activity in C8D30 cell-line when dCas9-VP64-gRNA complex is expressed by A1 reactive astrocyte specific promoters (pTimp1 and pSteap4).

2. pSynt

C8-D30 cells were co-transfected with Lenti viral vector: pTimp1-dCas9-VP64 and pSynt. dCas9-VP64-gRNA complex target and activate the pMLPm promoter in our design. This activation have driven pMLPm to regulate the expression of exogenous reverse caspase3 fused to mCherry fluorophore by T2A peptide (Fig 4).

Figure 4: Images of mCherry in C8-D30 cells 24 hours post co-transfection with lenti:pTimp1-dCas9-VP64 and pSynt. A – Bright field. B – Fluorescent microscope

mCherry expression in C8-D30 reactive astrocyte cells was visible. Demonstrating the activity of our dCas9 editing approach and verifying expression of reverse caspase3. In fluorescent microscope we were able to detect mCherry fluorophore in several cells.

By demonstrating mCherry expression in C8-D30 cell line we were able to demonstrate both the specificity of our chosen promoters (pTImp1 and pSteap4) as well as the ability of our system to drive the expression of caspase3 (through the mCherry marker) we have designed into our plasmid.

References

1. Farzadfard, Fahim, Samuel D. Perli, and Timothy K. Lu. "Tunable and multifunctional eukaryotic transcription factors based on CRISPR/Cas." ACS synthetic biology 2.10 (2013): 604-613.‏

2. La Russa, Marie F., and Lei S. Qi. "The new state of the art: CRISPR for gene activation and repression." Molecular and cellular biology (2015): MCB-00512.‏

3. Kim, Jin Hee, et al. "High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice." PloS one 6.4 (2011): e18556.‏

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 8
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 178
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]