Difference between revisions of "Part:BBa K1699002"

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<partinfo>BBa_K1699002 short</partinfo>
 
<partinfo>BBa_K1699002 short</partinfo>
  
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Ribozyme flanked gRNA compatible for SaCas9. gRNA sequence complements 3 different loci in the second exon of human UBB gene. It has a hammerhead ribozyme on its 5' and an HDV ribozyme on its 3' end. Upon transcription the ribozymes should cleave the mRNA at specific locations to release the gRNA (6).  
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Ribozyme flanked gRNA compatible for SaCas9 for gene knock-out. gRNA sequence is complementary to 3 different loci in the second exon of human Ubiquitin B (UBB) gene, which is essential for cancer cells (1, 2). 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 (3, 4).
  
 
===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 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).
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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. gRNA scaffold sequence for SaCas9 was used (5). In order to utilize the cancer-specific promoter hyperactivation, we used an RGR (Ribozyme gRNA Ribozyme) design (3, 4). 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. UBB gene was chosen as a model for SaCas9-mediated gene knock-out. UBB is essential for survival of multiple cancer cell types (1, 2).
  
 
===Characterization===
 
===Characterization===
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This part was used in a following construct:
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'''pSurvivin-gUBB''': AAV vector for the synthesis of gRNA for SaCas9 targeting human UBB gene (abbreviated as gUBB) 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 gene knock-out (the other being human TERT promoter ([https://parts.igem.org/Part:BBa_K1699001]) for a control of SaCas9).
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[[Image:PSurvivin-gUBB-polyA-pAAV_Map.jpg|center|500px|thumb|'''Fig. 1'''. Plasmid map of AAV vector carrying gUBB under the control of human survivin promoter.]]
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The design of cancer-specific CRISPR-mediated gene knock-out is summarized in Fig. 2.
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[[Image:Knock-out_(full).jpg|center|500px|thumb|'''Fig. 2'''. Summary of two cancer-specific promoter-driven CRISPR-mediated gene knock-out.]]
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===References===
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1. Downregulation of ubiquitin level via knockdown of polyubiquitin gene Ubb as potential cancer therapeutic intervention.
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Oh C, Park S, Lee EK, Yoo YJ.
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Sci Rep. 2013;3:2623. doi: 10.1038/srep02623. http://www.ncbi.nlm.nih.gov/pubmed/24022007
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2. The ubiquitin-proteasome system as a prospective molecular target for cancer treatment and prevention.
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Chen D, Dou QP.
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Curr Protein Pept Sci. 2010 Sep;11(6):459-70. Review. http://www.ncbi.nlm.nih.gov/pubmed/20491623
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3. 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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4. 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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5. In vivo genome editing using Staphylococcus aureus Cas9. Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, Zetsche B, Shalem O, Wu X, Makarova KS, Koonin EV, Sharp PA, Zhang F. Nature. 2015 Apr 9;520(7546):186-91. doi: 10.1038/nature14299. Epub 2015 Apr 1. http://www.ncbi.nlm.nih.gov/pubmed/25830891
  
  

Latest revision as of 13:44, 13 September 2015

gRNA for SaCas9 targeting human ubiquitin B gene

Ribozyme flanked gRNA compatible for SaCas9 for gene knock-out. gRNA sequence is complementary to 3 different loci in the second exon of human Ubiquitin B (UBB) gene, which is essential for cancer cells (1, 2). 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 (3, 4).

Usage and Biology

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. gRNA scaffold sequence for SaCas9 was used (5). In order to utilize the cancer-specific promoter hyperactivation, we used an RGR (Ribozyme gRNA Ribozyme) design (3, 4). 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. UBB gene was chosen as a model for SaCas9-mediated gene knock-out. UBB is essential for survival of multiple cancer cell types (1, 2).

Characterization

This part was used in a following construct:

pSurvivin-gUBB: AAV vector for the synthesis of gRNA for SaCas9 targeting human UBB gene (abbreviated as gUBB) 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 gene knock-out (the other being human TERT promoter ([1]) for a control of SaCas9).

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

The design of cancer-specific CRISPR-mediated gene knock-out is summarized in Fig. 2.

Fig. 2. Summary of two cancer-specific promoter-driven CRISPR-mediated gene knock-out.

References

1. Downregulation of ubiquitin level via knockdown of polyubiquitin gene Ubb as potential cancer therapeutic intervention. Oh C, Park S, Lee EK, Yoo YJ. Sci Rep. 2013;3:2623. doi: 10.1038/srep02623. http://www.ncbi.nlm.nih.gov/pubmed/24022007

2. The ubiquitin-proteasome system as a prospective molecular target for cancer treatment and prevention. Chen D, Dou QP. Curr Protein Pept Sci. 2010 Sep;11(6):459-70. Review. http://www.ncbi.nlm.nih.gov/pubmed/20491623

3. 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

4. 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

5. In vivo genome editing using Staphylococcus aureus Cas9. Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, Zetsche B, Shalem O, Wu X, Makarova KS, Koonin EV, Sharp PA, Zhang F. Nature. 2015 Apr 9;520(7546):186-91. doi: 10.1038/nature14299. Epub 2015 Apr 1. http://www.ncbi.nlm.nih.gov/pubmed/25830891


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 146
    Illegal NgoMIV site found at 175
  • 1000
    COMPATIBLE WITH RFC[1000]