Part:BBa_K3017066
Construct for testing CRISPRi asRNA de-suppression effect of CRISPRi sgRNA
The asRNA characterization construct is designed to prove asRNA ability to derepress a CRISPRi effect under arabinose induction with pBAD in vivo.
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 GFP, 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 all sgRNA transcription, weak Anderson promoter BBa_J23115 of strength 0.15 is used to allow for faster response rate between repressed state to derepressed state after arabinose induction.
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 inStreptococcus 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
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 3350
Illegal PstI site found at 4772
Illegal PstI site found at 4976
Illegal PstI site found at 5006
Illegal PstI site found at 6218 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 933
Illegal NheI site found at 956
Illegal NheI site found at 2326
Illegal NheI site found at 2506
Illegal NheI site found at 2529
Illegal PstI site found at 3350
Illegal PstI site found at 4772
Illegal PstI site found at 4976
Illegal PstI site found at 5006
Illegal PstI site found at 6218 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2811
Illegal BamHI site found at 2265 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 3350
Illegal PstI site found at 4772
Illegal PstI site found at 4976
Illegal PstI site found at 5006
Illegal PstI site found at 6218 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 3350
Illegal PstI site found at 4772
Illegal PstI site found at 4976
Illegal PstI site found at 5006
Illegal PstI site found at 6218
Illegal NgoMIV site found at 3638
Illegal NgoMIV site found at 4742
Illegal NgoMIV site found at 4815
Illegal NgoMIV site found at 5300
Illegal NgoMIV site found at 6209
Illegal AgeI site found at 2100 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 705
Illegal SapI site found at 2082
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