Team:BGU Israel/Design
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Team:BGU Israel
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Detailed design
Design 1: Cancer-specific CRISPR/Cas9-mediated gene knock-out
This design utilizes “classical” CRISPR-Cas9 system for knock-out of a cancer-essential gene.
The system includes two parts: one AAV expression cassette with the Cas9 gene, and the other with a gRNA designed to target 3 sequence repeats in the second exon of Ubb.
Cas9 – the Cas9 endonuclease was assembled into an expression vector under hTERT promoter. Therefore Cas9 should be expressed predominantly in cells in which the promoter is highly active, namely – cancer cells (1). We utilized Staphylococcus aureus Cas9 version (SaCas9) (4).
gRNA – the guide RNA is a hundred base-long molecule with a unique two dimensional structure which binds Cas9 and guides it to a dsDNA sequence complementary to 21-22 base pairs on the 5' end of the molecule. The gRNA was also assembled into a AAV vector, under the control of human survivin promoter. gRNA scaffold sequence for SaCas9 was used (5). In order to utilize the cancer specific promoter hyperactivation we used an RGR (Ribozyme gRNA Ribozyme). This design allows for gRNAs to be transcribed and processed using RNA polymerase II promoters, since these are the main promoters controlling gene activation (6).
When both conditions are met, meaning the system is in a cancer cell in which both promoters are highly active, the SaCas9 is guided by the gRNA to the target DNA, and introduces double strand breaks (DSB) at the target site. This then leads to activation of intrinsic DNA damage repair mechanism - predominantly error-prone non-homologous end joining (NHEJ), which introduces insertion/deletion mutations. This, in turn, can significantly disrupt a coding sequence, eliminating partially or completely a target protein function.
For a proof-of-concept of the knock-out system, we chose Ubiquitin B (Ubb) gene which encodes for poly-Ubiquitin, as a target for gRNA-guided SaCas9. Ubiquitin levels are elevated in most, if not all human cancer cells, it is essential to the growth of cancer cells, and the protein product of the gene is thought to help cancer cells adapt to increased stress (7). Ubb emerges as one of the promising targets for cancer therapy. For example, Ubb downregulation by siRNA has shown a high decrease in tumor proliferation and increased apoptosis, both in vitro and in vivo (7).
Design 2: Cancer-specific CRISPR/Cas9-mediated activation of the gene of interest
Take updated figure
This design utilizes modified CRISPR-Cas9 system for transcriptional activation of any gene of interest.
The system includes 3 parts: one AAV expression cassette with the activator Cas9 (dCas9-VP64), the second with a gRNA designed to guide activator Cas9 to synthetic promoter, and the third with the gene of interest (modelled by GFP) under the control of synthetic activation promoter.
dCas9-VP64 – dCas9-VP64 was engineered so that it lacks endonuclease activity (“dead” Cas9) and has 4 VP16 activation domains fused to the protein. When guided to a specific promoter, dCas9-VP64 activates transcription of genes downstream of its binding site (8). dCas9-VP64 was assembled into an expression vector under hTERT promoter.
gRNA- The gRNA (using the scaffold for Staphylococcus pyogenes Cas9) was also assembled into a AAV vector, under the control of human survivin promoter, using previously described ribozyme design. The gRNA sequence is used to guide dCas9-V64 to a specific synthetic promoter.
Synthetic activation promoter- The third part of the system is an expression AAV cassette with GFP under the control of synthetic promoter (9). The synthetic promoter has 3 complementary sites for the gRNA, which, upon binding of dCas9-VP64 in a tandem, should promote transcription of a downstream gene.
For a proof-of-concept, we utilized GFP as our target gene. The design allows for an expression of any desired protein: 1) to induce cancer cell apoptosis or cell death by using reversed caspase-3, which can lead to apoptosis when expressed in cells, or diphteria toxin A, which can kill a cell (10,11), 2) label the tumor for complete surgical removal by using chromoproteins; and 3) produce a biomarker detectable in the blood or urine, for cancer diagnosis, for example, by using SEAP (Secreted embryonic alkaline phosphatase), which can be excreted out of the cells and its levels monitored easily (12) (figure 3).
| Figure 3 - |
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Detailed design and cloning program
1) Design of “master” template with specific restriction sites for subcloning
2) Design of Boomerang components Our basic components include promoters, Cas9 proteins and guideRNAs.
BLASTTT
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Synthesized Components |
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Name |
Description |
Source |
Map, Sequence |
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dCas9-VP64 |
Activator Cas9 for promotion of exogenous genes
expression |
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SaCas9 |
“Classical” Cas9 endonuclease for knock-down of
target genes |
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gMLP |
Ribozyme flanked guide RNA leading dCas9-VP64
to the synthetic promoter |
IDT |
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gUBB |
Ribozyme flaked guide RNA leading Cas9 to cut
UBB gene |
IDT |
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U6 |
As positive control for RGR design |
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pSurvivin |
Promoter for survivin
gene |
Synthezza Bioscience |
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phTERT |
Promoter for hTERT
gene |
Synthezza Bioscience |
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hTERT eGFP poly A - MASTER |
Master. Cloned into delivery vector as a
cloning template for all other inserts. |
Synthezza Bioscience |
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3. Cloning of “master” into AAV vector
4. Cloning of various Boomerang components into “MASTER-AAV”
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Activation System |
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Name |
Description |
Source |
Map, Sequence |
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phTERT-dCas9-VP64-polyA-pAAV |
dCas9-VP64 under hTERT
promoter. System part. |
Synthezza Bioscience |
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CMV-dCas9-VP64-polyA-pAAV |
dCas9 under CMV promoter. Positive control for
dCas9. |
Self cloning |
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pSurvivin-gMLP-polyA-pAAV |
Ribozyme flanked gRNA
for the synthetic promoter under Survivin promoter.
System part. |
Synthezza Bioscience |
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U6-gMLP-pAAV |
gRNA for the synthetic promoter under U6 promoter.
Positive control for gMLP. |
Self cloning |
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pMLPm-eGFP-polyA-pAAV |
GFP under the synthetic promoter. System part. |
Synthezza Bioscience |
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Knock-out System |
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Name |
Description |
Source |
Map, Sequence |
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phTERT-SaCas9-polyA-pAAV |
SaCas9 under hTERT
promoter. |
Synthezza Bioscience |
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CMV-SaCas9-polyA-pAAV |
SaCas9 under CMV. Positive control for SaCas9. |
Self cloning |
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pSurvivin-gUBB-polyA-pAAV |
gRNA for UBB gene under Survivin.
System part. |
Synthezza Bioscience |
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U6-gUBB-pAAV |
gRNA for UBB gene under U6 promoter. Control for
RGR design and gUBB. |
Self cloning |
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General Controls |
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Name |
Description |
Source |
Map, Sequence |
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phTERT-eGFP-polyA master-pAAV |
GFP under hTERT
promoter. Positive control for hTERT. |
Synthezza Bioscience |
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pSurvivin-mCherry-polyA-pAAV |
mCherry under Survivin
promoter. Positive control for Survivin. |
Self cloning |
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References
(1) The telomerase reverse transcriptase promoter drives efficacious tumor suicide gene therapy while preventing hepatotoxicity encountered with constitutive promoters
(2) Applications of the CRISPR–Cas9 system in cancer biology
(3) Oncolytic viruses: a new class of immunotherapy drugs
(4) Targeting of tumor radioiodine therapy by expression of the sodium iodide symporter under control of the survivin promoter
(5) In vivo genome editing using Staphylococcus aureus Cas9.
(6) Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing
(7) DDownregulation of ubiquitin level via knockdown of polyubiquitin gene Ubb as potential cancer therapeutic intervention
(8) RNA-guided gene activation by CRISPR-Cas9-based transcription factors.
(9) Tunable and Multifunctional Eukaryotic Transcription Factors Based on CRISPR/Cas
(10) Generation of Constitutively Active Recombinant Caspases-3 and -6 by Rearrangement of Their Subunits
(11) One molecule of diphtheria toxin fragment a introduced into a cell can kill the cell
(12) SEAP expression in transiently transfected mammalian cells grown in serum-free suspension culture
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