pSteap4 RGR gRNA
pSteap4 (Six Transmembrane Epithelial Antigen of Prostate 4 promoter) is regulating the expression of Six Transmembrane Epithelial Antigen of Prostate 4 gene which is highly expressed in A1 reactive astrocyte cells. [1,2]
In this composite part this promoter regulates the expression of gRNA which its spacer is complementary to three different loci in the synthetic minimal adenovirus major late promoter (pMLPm). The promoter will be active only under the condition that gRNA-dCas9-VP64 complex promotes downstream transcription. When the transcription factor VP64 (an engineered tetramer of the herpes simplex virus 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. 
The gRNA is flanked with hammerhead ribozyme on the 5' and HDV ribozyme on the 3' (RGR), the system will undergo self-catalyzed cleavage after translation to generate the mature gRNA structure. This will enable the gRNA to be expressed not only by RNA polymerase promoter III like U6, but also by RNA polymerase II promoter, meaning we could use specific promoters that will be active only in our target cells, such as pSteap4. [5,6]
Usage and Biology
Guide RNA (gRNA) is a hundred base-long molecule, it includes a scaffold sequence which has a unique two dimensional structure necessary for dCas9 binding and a spacer sequence designed to target the synthetic promoter pMLPm. gRNA scaffold sequence for staphylococcus aureus Cas9 (SaCas9 ) was used.
Deactivated Cas9 (dCas9) attached to transcription factors such as VP64 can be guided to a specific location in the genome to induce gene expression.
In ALS disease, under gliosis conditions astrocyte cells are transformed from their resting to their reactive form, this transformation includes the induction of several genes including Six Transmembrane Epithelial Antigen of Prostate 4 gene by pSteap4.[1,2]
In order to activate our CRISPR dCas9 VP64 system in a specific manner, only in reactive astrocytes and not in the resting cells, we used reactive astrocyte specific genes promoters to express the dCas9 VP64 and the gRNA. Since these specific promoters are RNA polymerase II promoters and not RNA polymerase III promoter like U6 the RGR will enable the gRNA expression. Here the gRNA is expressed by the pSteap4 promoter. The gRNA-dCas9-VP64 complex will target the pMLPm synthetic promoter and promote the expression of exogenous reverse caspase3, that in contrast to the endogenous caspas3 will be activated after transcription due to its autocatalytic processing, meaning this enzyme will trigger an apoptotic signal that will lead to apoptotic death (caspase3 is responsible for chromatin condensation and DNA fragmentation) . 
mCherry reporter protein is also expressed by the pMLPm promoter, it is fused to the exogenous reverse caspase3 with Thoseaasigna virus 2A (T2A) peptide that is cleaved during translation.
We will use the pSteap4 promoter to specifically express gRNA that will connect to dCas9 enzyme and VP64 transcription factors, together our system will target and activate the synthetic promoter pMLPm that will express reverse Caspase3, by doing so we will be able to induce apoptotic cell death only in reactive astrocyte and will discriminate against resting cells. By reducing the amount of reactive astrocytes we will prevent motor neuron death and prolong ALS patient survival.
Reactivity test by immunostaining of astrocyte cell-line C8-D30 (Conducted for us by: Dinorah Friedmann-Morvinski's lab on our cells)
Aiming for expression under the Steap4 promoter specifically in reactive astrocytes, we wanted to validate whether the cell-line that is used in our experiments, C8-D30, is indeed a good model for A1 reactive astrocytes. Reactivity confirmation was achieved via immuno-staining of known and established markers of reactive astrocytes (Fig 1).
High expression of GFAP and Nestin proteins is a specific feature of reactive astrocytes, which usually appears after various brain injuries . As can be seen in Figure 1, C8-D30 astrocytes cells displayed strong immunostaining signal for both GFAP and Nestin. Therefore, it is possible to conclude that the chosen cell-line of C8-D30, is indeed that of reactive astrocytes. It can serve as an accurate model for testing this project’s hypothesis, and to examine the specificity of pSteap4 promoter.
Gene expression due to pSteap4 RGR gRNA activity This part was used by the BGU 2018 team in pSynt construct.
In this construct pSteap4 regulates the expression of RGR gRNA. In our design the chosen gRNA spacer is complementary to three different loci in the pMLPm promoter.
C8-D30 cells were co-transfected with a Lenti-viral-vector: pTimp1-dCas9-VP64 and pSynt. dCas9-VP64-gRNA complex targets and activates the pMLPm promoter in our design, otherwise pMLPm promoter remain non-active . This activation caused pMLPm to regulate the expression of exogenous reverse caspase3 fused to mCherry fluorophore by T2A peptide (Fig 2).
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 we were able to demonstrate both the specificity of our chosen promoter as well as the ability of our system to drive the expression of caspase3, through the mCherry marker, that we have designed into our plasmid.
1. Liddelow, Shane A., et al. "Neurotoxic reactive astrocytes are induced by activated microglia." Nature 541.7638 (2017): 481.
2. Zamanian J, Xu L, Foo L, et al. Genomic Analysis of Reactive Astrogliosis. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2012;32(18):6391-6410. doi:10.1523/JNEUROSCI.6221-11.2012.
3. 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.
4. 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.
5. Nissim, Lior, et al. "Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells." Molecular cell 54.4 (2014): 698-710.
6. Gao, Yangbin, and Yunde Zhao. "Self‐processing of ribozyme‐flanked RNAs into guide RNAs in vitro and in vivo for CRISPR‐mediated genome editing." Journal of integrative plant biology56.4 (2014): 343-349.
7. Ran, F. Ann, et al. "In vivo genome editing using Staphylococcus aureus Cas9." Nature 520.7546 (2015): 186. 8. Perez-Pinera, Pablo, et al. "RNA-guided gene activation by CRISPR-Cas9–based transcription factors." Nature methods10.10 (2013): 973.
9. Srinivasula, Srinivasa M., et al. "Generation of constitutively active recombinant caspases-3 and-6 by rearrangement of their subunits." Journal of Biological Chemistry 273.17 (1998): 10107-10111.
10. 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.
11. Sosunov, Alexander A., et al. "Phenotypic conversions of “protoplasmic” to “reactive” astrocytes in Alexander disease." Journal of Neuroscience 33.17 (2013): 7439-7450.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25Illegal NgoMIV site found at 1077
Illegal NgoMIV site found at 1106
- 1000COMPATIBLE WITH RFC