Difference between revisions of "Part:BBa K1699003"

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===Usage and Biology===
 
===Usage and Biology===
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Guide RNA is a hundred base-long molecule with a unique two dimensional structure which binds dCas9-VP64 and guides it to a dsDNA sequence complementary to 21-22 base pairs on the 5' end of the molecule. Upon binding to promoter, dCas9-VP64-gRNA complex will promote transcription of genes downstream of binding site. gRNA scaffold sequence for SpdCas9-VP64 was used (4). In order to utilize the cancer-specific promoter hyperactivation, we used an RGR (Ribozyme gRNA Ribozyme) design (2, 3). This design allows for gRNAs to be transcribed and processed using RNA polymerase II promoters, since these are the main promoters controlling gene activation, while eliminating the need for use of constitutuve RNA Polymerase III promoters, like U6 promoter, which is generally used to synthesize gRNAs.
+
Guide RNA is a hundred base-long molecule with a unique two dimensional structure which binds dCas9-VP64 and guides it to a dsDNA sequence complementary to 21-22 base pairs on the 5' end of the molecule. Upon binding to promoter, dCas9-VP64-gRNA complex will promote transcription of genes downstream of binding site. gRNA scaffold sequence for Sp dCas9-VP64 was used (4). In order to utilize the cancer-specific promoter hyperactivation, we used an RGR (Ribozyme gRNA Ribozyme) design (2, 3). This design allows for gRNAs to be transcribed and processed using RNA polymerase II promoters, since these are the main promoters controlling gene activation, while eliminating the need for use of constitutive RNA Polymerase III promoters, like U6 promoter, which is generally used to synthesize gRNAs.
  
 
===Characterization===
 
===Characterization===
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This part has been validated by transfection of human cancer cells HepG2 with dCas9-VP64, this part, and a synthetic promoter with three matching sequences for the gRNA, upstream of GFP. Successfull expression of GFP was observed (Figure 1).
 
  
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Figure 1
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This part was used and validated by BGU 2015 team in a following construct:
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'''pSurvivin-gMLP''': AAV vector for the synthesis of gRNA for dCas9-VP64 targeting synthetic activation promoter pMLPm (abbreviated as gMLP) under the control of human survivin promoter (Fig. 1). The survivin promoter was used as a part of two cancer-specific promoter-based design for CRISPR-mediated transcriptional activation system (the other being human TERT promoter ([https://parts.igem.org/Part:BBa_K1699001]) for a control of dCas9-VP64).
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[[Image:PSurvivin-gMLP-polyA-pAAV_Map.jpg|center|500px|thumb|'''Fig. 1'''. Plasmid map of AAV vector carrying gMLP under the control of human survivin promoter.]]
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Following simultaneous plasmid transfection of dCas9-VP64 - under the control of hTERT promoter, gMLP (targeting the synthetic activation promoter) - under the control of human survivin promoter, and eGFP under synthetic activation promoter (1), eGFP expression was detected only in cancer cells, compared to undetected levels in healthy cells (Fig. 2).
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[[Image:Activation.jpg|center|500px|thumb|'''Fig. 2'''. eGFP expression from synthetic activation promoter exclusively in cancer cells after successful activation of CRISPR-based activation core driven by dCas9-VP64 - under the control of hTERT promoter, and gMLP (targeting the synthetic activation promoter) - under the control of human survivin promoter. Bar:100 micron.]]
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The expression of dCas9-VP64 and gRNA under the control of cancer-specific promoters (TERT and survivin) drives the activation of the system only in cancer cells (Fig. 3).
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[[Image:Activ2(full).jpg|center|500px|thumb|'''Fig. 3'''. Cancer-specific CRISPR-based activation of the gene of interest using two cancer-specific promoter-driven expression of dCas9-VP64 and gRNA.]]
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===References===
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1. Tunable and multifunctional eukaryotic transcription factors based on CRISPR/Cas. Farzadfard F, Perli SD, Lu TK. ACS Synth Biol. 2013 Oct 18;2(10):604-13. doi: 10.1021/sb400081r. Epub 2013 Sep 11. http://www.ncbi.nlm.nih.gov/pubmed/23977949
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 +
2. Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing. Gao Y, Zhao Y. J Integr Plant Biol. 2014 Apr;56(4):343-9. doi: 10.1111/jipb.12152. Epub 2014 Mar 6. http://www.ncbi.nlm.nih.gov/pubmed/24373158
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 +
3. Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells. Nissim L, Perli SD, Fridkin A, Perez-Pinera P, Lu TK. Mol Cell. 2014 May 22;54(4):698-710. doi: 10.1016/j.molcel.2014.04.022. Epub 2014 May 15. http://www.ncbi.nlm.nih.gov/pubmed/24837679
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 +
4. RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Perez-Pinera P, Kocak DD, Vockley CM, Adler AF, Kabadi AM, Polstein LR, Thakore PI, Glass KA, Ousterout DG, Leong KW, Guilak F, Crawford GE, Reddy TE, Gersbach CA. Nat Methods. 2013 Oct;10(10):973-6. doi: 10.1038/nmeth.2600. Epub 2013 Jul 25. http://www.ncbi.nlm.nih.gov/pubmed/23892895
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Latest revision as of 12:12, 14 September 2015

gRNA for dCas9-VP64 targeting synthetic activation promoter pMLPm

Ribozyme flanked gRNA compatible for dCas9-VP64. gRNA sequence is complementary to 3 different loci in the synthetic promoter pMLPm (1), and gRNA-dCas9-VP64 complex can promote transcription downstream of synthetic promoter. It has a hammerhead ribozyme on its 5' and an HDV ribozyme on its 3' end. Upon transcription the ribozymes cleave the RNA at specific locations to release the mature gRNA (2, 3).


Usage and Biology

Guide RNA is a hundred base-long molecule with a unique two dimensional structure which binds dCas9-VP64 and guides it to a dsDNA sequence complementary to 21-22 base pairs on the 5' end of the molecule. Upon binding to promoter, dCas9-VP64-gRNA complex will promote transcription of genes downstream of binding site. gRNA scaffold sequence for Sp dCas9-VP64 was used (4). In order to utilize the cancer-specific promoter hyperactivation, we used an RGR (Ribozyme gRNA Ribozyme) design (2, 3). This design allows for gRNAs to be transcribed and processed using RNA polymerase II promoters, since these are the main promoters controlling gene activation, while eliminating the need for use of constitutive RNA Polymerase III promoters, like U6 promoter, which is generally used to synthesize gRNAs.

Characterization

This part was used and validated by BGU 2015 team in a following construct:

pSurvivin-gMLP: AAV vector for the synthesis of gRNA for dCas9-VP64 targeting synthetic activation promoter pMLPm (abbreviated as gMLP) under the control of human survivin promoter (Fig. 1). The survivin promoter was used as a part of two cancer-specific promoter-based design for CRISPR-mediated transcriptional activation system (the other being human TERT promoter ([1]) for a control of dCas9-VP64).

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Fig. 1. Plasmid map of AAV vector carrying gMLP under the control of human survivin promoter.

Following simultaneous plasmid transfection of dCas9-VP64 - under the control of hTERT promoter, gMLP (targeting the synthetic activation promoter) - under the control of human survivin promoter, and eGFP under synthetic activation promoter (1), eGFP expression was detected only in cancer cells, compared to undetected levels in healthy cells (Fig. 2).

Fig. 2. eGFP expression from synthetic activation promoter exclusively in cancer cells after successful activation of CRISPR-based activation core driven by dCas9-VP64 - under the control of hTERT promoter, and gMLP (targeting the synthetic activation promoter) - under the control of human survivin promoter. Bar:100 micron.

The expression of dCas9-VP64 and gRNA under the control of cancer-specific promoters (TERT and survivin) drives the activation of the system only in cancer cells (Fig. 3).

Fig. 3. Cancer-specific CRISPR-based activation of the gene of interest using two cancer-specific promoter-driven expression of dCas9-VP64 and gRNA.

References

1. Tunable and multifunctional eukaryotic transcription factors based on CRISPR/Cas. Farzadfard F, Perli SD, Lu TK. ACS Synth Biol. 2013 Oct 18;2(10):604-13. doi: 10.1021/sb400081r. Epub 2013 Sep 11. http://www.ncbi.nlm.nih.gov/pubmed/23977949

2. Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing. Gao Y, Zhao Y. J Integr Plant Biol. 2014 Apr;56(4):343-9. doi: 10.1111/jipb.12152. Epub 2014 Mar 6. http://www.ncbi.nlm.nih.gov/pubmed/24373158

3. Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells. Nissim L, Perli SD, Fridkin A, Perez-Pinera P, Lu TK. Mol Cell. 2014 May 22;54(4):698-710. doi: 10.1016/j.molcel.2014.04.022. Epub 2014 May 15. http://www.ncbi.nlm.nih.gov/pubmed/24837679

4. RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Perez-Pinera P, Kocak DD, Vockley CM, Adler AF, Kabadi AM, Polstein LR, Thakore PI, Glass KA, Ousterout DG, Leong KW, Guilak F, Crawford GE, Reddy TE, Gersbach CA. Nat Methods. 2013 Oct;10(10):973-6. doi: 10.1038/nmeth.2600. Epub 2013 Jul 25. http://www.ncbi.nlm.nih.gov/pubmed/23892895


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 144
    Illegal NgoMIV site found at 173
  • 1000
    COMPATIBLE WITH RFC[1000]