Difference between revisions of "Part:BBa K3017067"
 
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<partinfo>BBa_K3017067 short</partinfo>
 
<partinfo>BBa_K3017067 short</partinfo>
  
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<H2>Aim</H2>
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<P>This asRNA characterization construct is to characterize the cross-talking by sgRNA mismatch with different FP.</P>
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<P>This asRNA characterization construct is to characterize the cross talking by sgRNA mismatch with different FP.</P>
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<p>asRNA is a purely synthetic RNA that is designed to bind with its paired sgRNA at its extensor region. By removing the secondary structure of sgRNA extensor, dCas9 leave the sgRNA. The CRISPRi repression is reversed by the asRNA.[1]</p>
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<H2>Design</H2>
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<P>The asRNA characterization constructs all contain a constitutively expressed dCas9, a fluorescent protein RFP, asRNA under pBAD promoter, and sgRNA (GFP). Under the regulation of pBAD promoter, asRNA is transcripted when arabinose is added to the culture medium. The transcription start site of the pBAD promoter has been identified according to Brzozowska et al. (2018), in which the asRNA has been placed on the Transcription Start Site (TSS). Forl sgRNA transcription, weak Anderson promoter BBa_J23115 of strength 0.15 is used. Since the sgRNA transcribed will be targeting for for GFP, suppression effect on the RFP should be non-existing. This allows us to assess the cross talking by sgRNA mismatch.</P>
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<P>The asRNA characterization constructs all contain a constitutively expressed dCas9, a fluorescent protein RFP, asRNA under pBAD promoter, and sgRNA (GFP). Under the regulation of pBAD promoter, asRNA is transcripted when arabinose is added to the culture medium. The transcription start site of the pBAD promoter has been identified according to Brzozowska et al. (2018), in which the asRNA has been placed on the Transcription Start Site (TSS). For sgRNA transcription, weak Anderson promoter BBa_J23115 of strength 0.15 is used. Since the sgRNA transcribed will be targeting for <i>gfp</i>, suppression effect on the RFP should be non-desirable. This allows us to assess the cross-talking by sgRNA mismatch.</P>
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<br>
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<p><b>References:</b></p>
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<br>[1] Y. J. Lee, A. Hoynes-Oconnor, M. C. Leong, and T. S. Moon, “Programmable control of bacterial gene expression with the combined CRISPR and antisense RNA system,” Nucleic Acids Research, vol. 44, no. 5, pp. 2462–2473, Feb. 2016.
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<br>[2] C. Anders, O. Niewoehner, A. Duerst, and M. Jinek, “Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease,” Nature, vol. 513, no. 7519, pp. 569–573, 2014.
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<br>[3] S. H. Sternberg, S. Redding, M. Jinek, E. C. Greene, and J. A. Doudna, “DNA Interrogation by the CRISPR RNA-Guided Endonuclease Cas9,” Biophysical Journal, vol. 106, no. 2, 2014.
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<br>[4] T. Karvelis, G. Gasiunas, A. Miksys, R. Barrangou, P. Horvath, and V. Siksnys, “crRNA and tracrRNA guide Cas9-mediated DNA interference in Streptococcus thermophilus,” RNA Biology, vol. 10, no. 5, pp. 841–851, 2013.
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<br>[5] T. Møller, T. Franch, P. Højrup, D. R. Keene, H. P. Bächinger, R. G. Brennan, and P. Valentin-Hansen, “Hfq,” Molecular Cell, vol. 9, no. 1, pp. 23–30, 2002.
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<br>[6] G. M. Cech, A. Szalewska-Pałasz, K. Kubiak, A. Malabirade, W. Grange, V. Arluison, and G. Węgrzyn, “The Escherichia Coli Hfq Protein: An Unattended DNA-Transactions Regulator,” Frontiers in Molecular Biosciences, vol. 3, 2016.
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<br>[7]N. Brzozowska et al., “Characterizing Genetic Circuit Components in E. coli towards a Campylobacter jejuni Biosensor,” p. 290155, 2018.
  
  

Latest revision as of 01:55, 22 October 2019


Construct for testing CRISPRi sgRNA cross-talk with non-target DNA

This asRNA characterization construct is to characterize the cross-talking by sgRNA mismatch with different FP.

asRNA is a purely synthetic RNA that is designed to bind with its paired sgRNA at its extensor region. By removing the secondary structure of sgRNA extensor, dCas9 leave the sgRNA. The CRISPRi repression is reversed by the asRNA.[1]

The asRNA characterization constructs all contain a constitutively expressed dCas9, a fluorescent protein RFP, asRNA under pBAD promoter, and sgRNA (GFP). Under the regulation of pBAD promoter, asRNA is transcripted when arabinose is added to the culture medium. The transcription start site of the pBAD promoter has been identified according to Brzozowska et al. (2018), in which the asRNA has been placed on the Transcription Start Site (TSS). For sgRNA transcription, weak Anderson promoter BBa_J23115 of strength 0.15 is used. Since the sgRNA transcribed will be targeting for gfp, suppression effect on the RFP should be non-desirable. This allows us to assess the cross-talking by sgRNA mismatch.



References:


[1] Y. J. Lee, A. Hoynes-Oconnor, M. C. Leong, and T. S. Moon, “Programmable control of bacterial gene expression with the combined CRISPR and antisense RNA system,” Nucleic Acids Research, vol. 44, no. 5, pp. 2462–2473, Feb. 2016.
[2] C. Anders, O. Niewoehner, A. Duerst, and M. Jinek, “Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease,” Nature, vol. 513, no. 7519, pp. 569–573, 2014.
[3] S. H. Sternberg, S. Redding, M. Jinek, E. C. Greene, and J. A. Doudna, “DNA Interrogation by the CRISPR RNA-Guided Endonuclease Cas9,” Biophysical Journal, vol. 106, no. 2, 2014.
[4] T. Karvelis, G. Gasiunas, A. Miksys, R. Barrangou, P. Horvath, and V. Siksnys, “crRNA and tracrRNA guide Cas9-mediated DNA interference in Streptococcus thermophilus,” RNA Biology, vol. 10, no. 5, pp. 841–851, 2013.
[5] T. Møller, T. Franch, P. Højrup, D. R. Keene, H. P. Bächinger, R. G. Brennan, and P. Valentin-Hansen, “Hfq,” Molecular Cell, vol. 9, no. 1, pp. 23–30, 2002.
[6] G. M. Cech, A. Szalewska-Pałasz, K. Kubiak, A. Malabirade, W. Grange, V. Arluison, and G. Węgrzyn, “The Escherichia Coli Hfq Protein: An Unattended DNA-Transactions Regulator,” Frontiers in Molecular Biosciences, vol. 3, 2016.
[7]N. Brzozowska et al., “Characterizing Genetic Circuit Components in E. coli towards a Campylobacter jejuni Biosensor,” p. 290155, 2018.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 3336
    Illegal PstI site found at 4758
    Illegal PstI site found at 4962
    Illegal PstI site found at 4992
    Illegal PstI site found at 6204
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
    Illegal NheI site found at 919
    Illegal NheI site found at 942
    Illegal NheI site found at 2312
    Illegal NheI site found at 2492
    Illegal NheI site found at 2515
    Illegal PstI site found at 3336
    Illegal PstI site found at 4758
    Illegal PstI site found at 4962
    Illegal PstI site found at 4992
    Illegal PstI site found at 6204
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2797
    Illegal BamHI site found at 2251
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 3336
    Illegal PstI site found at 4758
    Illegal PstI site found at 4962
    Illegal PstI site found at 4992
    Illegal PstI site found at 6204
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 3336
    Illegal PstI site found at 4758
    Illegal PstI site found at 4962
    Illegal PstI site found at 4992
    Illegal PstI site found at 6204
    Illegal NgoMIV site found at 3624
    Illegal NgoMIV site found at 4728
    Illegal NgoMIV site found at 4801
    Illegal NgoMIV site found at 5286
    Illegal NgoMIV site found at 6195
    Illegal AgeI site found at 616
    Illegal AgeI site found at 728
    Illegal AgeI site found at 2086
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 2068