Difference between revisions of "Part:BBa K3017068"

Latest revision as of 01:55, 22 October 2019

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

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

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]

This asRNA characterization constructs all contain a constitutively expressed dCas9, a fluorescent protein RFP, asRNA (for sgRNA_GFP) under pBAD promoter, and sgRNA (RFP). 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 rfp suppression effect on the RFP should be observed. A matching asRNA should reverse the suppression. Therefore, upon L-arabinose induction for the transcription of asRNA, no derepression effect is expected to be observed as the asRNA’s extensor is not complementary to the sgRNA_RFP.

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