OriginALS

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OriginALS Astrocyte Microglia CRISPR Promoters

OriginALS collection

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with no available treatment. The BGU-IGEM team aims to develop a system that will ultimately prolong survival of ALS patients by targeting microglia and reactive astrocytes, which are both non-neuronal cells that directly contribute to motor neuronal damage.
The "OriginALS" collection was designed to fulfill our therapeutic approach:

Inhibition of toxic pro-inflammatory cytokines secretion in microglia cells.
Promoting intrinsic apoptotic signal in reactive astrocytes and preventing their toxic effect on motor neurons.

Short description	NickName	Part link
Tissue inhibitor of metalloproteinases-1 promoter (pTIMP1)	pTIMP1	BBa_K2538005
gRNA#1 for SaCas9 targeting of IKKβ under U6 promoter	U6 g1	BBa_K2538000
gRNA#2 for SaCas9 targeting of IKKβ under U6 promoter	U6-g2	BBa_K2538001
gRNA#5 for SaCas9 targeting of IKKβ under U6 promoter	U6-g5	BBa_K2538002
gRNA#6 for SaCas9 targeting of IKKβ under U6 promoter	U6-g6	BBa_K2538003
Microglia specific promoter pF4/80	pF4/80	BBa_K2538006
Six Transmembrane Epithelial Antigen of Prostate 4 promoter (pSTEAP4) with a point mutation	pSTEAP4 +mut	BBa_K2538007
Six Transmembrane Epithelial Antigen of Prostate 4 promoter (pSTEAP4)	pSTEAP4	BBa_K2538008
Synthetic minimal adenovirus major late promoter (pMLPm)	pMLP	BBa_K2538009
Ribozyme flanked gRNA for SadCas9 VP64 targeting synthetic activation promoter pMLPm	RGR gRNA	BBa_K2538010
pSTEAP4 RGR gRNA (composite part)	pSTEAP4 RGR gRNA	BBa_K2538011

Using modified genome editing technique, we built a system that specifically targets toxic astrocytes and prevents the formation of new ones which hopefully will slow down the progression of the disease. As the reactivity of microglia and astrocytes is common to many neurodegenerative diseases, our novel approach could be expanded to other neurodegenerative diseases.
In this unique collection you can find our specific promoters and gRNA.

Astrocyte collection

In the astrocytes pathway, we aim to identify and eliminate only reactive astrocytes. In order to identify the reactive astrocytes, we will use the promoters of the distinguishing markers, pSTEAP4 and pTIMP1. The TIMP1 and STEAP4 genes have high levels of expression specifically in A1 reactive astrocyte when compared to other cells in the central nervous system.
pTIMP1 promoter regulates the expression of dCas9 fused to VP64 transcription factor.
pSTEAP4 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 pMLPm 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 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.

Short description	NickName	Part link
Tissue inhibitor of metalloproteinases-1 promoter (pTIMP1)	pTIMP1	BBa_K2538005
Six Transmembrane Epithelial Antigen of Prostate 4 promoter (pSTEAP4) with a point mutation	pSTEAP4 +mut	BBa_K2538007
Six Transmembrane Epithelial Antigen of Prostate 4 promoter (pSTEAP4)	pSTEAP4	BBa_K2538008
Synthetic minimal adenovirus major late promoter (pMLPm)	pMLP	BBa_K2538009
Ribozyme flanked gRNA for SadCas9 VP64 targeting synthetic activation promoter pMLPm	RGR gRNA	BBa_K2538010
pSTEAP4 RGR gRNA (composite part)	pSTEAP4 RGR gRNA	BBa_K2538011

The gRNA-dCas9-VP64 complex will target the synthetic minimal adenovirus major late promoter (pMLPm) 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).
By reducing the amount of reactive astrocytes we will prevent motor neuron death and prolong ALS patient survival. In this collection you can find our specific A1 reactive astrocyte promoters (pSTEAP4 and pTIMP1), pMLPm promoter and gRNA targeting pMLPm promoter for activation via dCas9 VP64 system.

Microglia collection

In the microglia pathway, we will knockout the IKKβ gene in microglia cells, using the CRISPR-Cas9 system delivered by AAV6 virus that targets microglia cells. The IKKβ gene is transcribed and translated into a kinase which has a role as an activator upstream to the NFkB complex assembly. Therefore, the knockout will decrease the neuro-inflammation transduced by classical NFkB signal. Targeting specifically microglia cells, we chose to combine the pF4/80 promoter, which is a known macrophage marker, with AAV6 viral vector.

Short description	NickName	Part link
gRNA#1 for SaCas9 targeting of IKKβ under U6 promoter	U6 g1	BBa_K2538000
gRNA#2 for SaCas9 targeting of IKKβ under U6 promoter	U6-g2	BBa_K2538001
gRNA#5 for SaCas9 targeting of IKKβ under U6 promoter	U6-g5	BBa_K2538002
gRNA#6 for SaCas9 targeting of IKKβ under U6 promoter	U6-g6	BBa_K2538003
Microglia specific promoter pF4/80	pF4/80	BBa_K2538006

The pF4/80 promoter will be responsible for the expression of the Cas9 protein, while the U6 promoter, which is a non-specific promoter, is responsible for the expression of the IKKβ guide RNA sequence. Our CRISPR Cas9 system will target and knock out the IKKβ gene, resulting in the inhibition of pro-inflammatory cytokines synthesis and secretion and thus reduce the formation of new reactive astrocytes in the system. In this collection you can find pF4/80 promoter and 4 different gRNA targeting the IKKβ for gene knockout via CRISPR Cas9 system.

CRISPR gRNA Collection

Guide RNA (gRNA) is a hundred base-long molecule, it includes a scaffold sequence which has a unique two dimensional structure necessary for Cas9 binding and a spacer sequence designed to target a specific location in the genome.

In this collection you can find 4 different gRNA targeting the IKKβ for gene knockout via CRISPR Cas9 system, and gRNA for the synthetic minimal adenovirus major late promoter (pMLPm) activation via dCas9 VP64 system.

Short description	NickName	Part link
gRNA#1 for SaCas9 targeting of IKKβ under U6 promoter	U6 g1	BBa_K2538000
gRNA#2 for SaCas9 targeting of IKKβ under U6 promoter	U6-g2	BBa_K2538001
gRNA#5 for SaCas9 targeting of IKKβ under U6 promoter	U6-g5	BBa_K2538002
gRNA#6 for SaCas9 targeting of IKKβ under U6 promoter	U6-g6	BBa_K2538003
Ribozyme flanked gRNA for SadCas9 VP64 targeting synthetic activation promoter pMLPm	RGR gRNA	BBa_K2538010

In a future therapeutic approach, it is envisioned that all gRNAs variants will be used together in as a cocktail of viruses in order to achieve higher CRISPR Cas9 efficiency.

Promoters collection

In this collection you can find all the promoters we used in our project targeting different types of cells. pTIMP1 and pSTEAP4 promoters were chosen to target A1 reactive astrocyte, since the TIMP1 and STEAP4 genes have high levels of expression specifically in those cells when compared to other cells in the central nervous system. pF4/80 were chosen to target microglia cells.

Short description	NickName	Part link
Tissue inhibitor of metalloproteinases-1 promoter (pTIMP1)	pTIMP1	BBa_K2538005
Microglia specific promoter pF4/80	pF4/80	BBa_K2538006
Six Transmembrane Epithelial Antigen of Prostate 4 promoter (pSTEAP4) with a point mutation	pSTEAP4 +mut	BBa_K2538007
Six Transmembrane Epithelial Antigen of Prostate 4 promoter (pSTEAP4)	pSTEAP4	BBa_K2538008
Synthetic minimal adenovirus major late promoter (pMLPm)	pMLP	BBa_K2538009