Team:Hong Kong HKUST/Basic Part
Best New Basic Part: BBa_K3017001
This part, BBa_K3017001, and other single-guide RNA are designed with many references to this great study on “Programmable control of bacterial gene expression with the combined CRISPR and antisense RNA system” by authors Y. J. Lee, A. Hoynes-Oconnor, M. C. Leong, and T. S. Moon. The link to the study can be found here:
https://www.ncbi.nlm.nih.gov/pubmed/26837577
dCas9 protein is directed to the specific DNA locus by a single-guide RNA (sgRNA), where it binds to suppress downstream gene expression. With reference to the research [1] on reversible CRISPRi switch, we redesigned the traditional sgRNA by adding an artificial linker behind crRNA and tracrRNA and modified the 3-component-sgRNA to suit our suppression purpose. Our design of sgRNA is compatible with spCas9.
Secondary structure of sgRNA is predicted by a software, NUPACK
Spacer - crRNA
crisprRNA(crRNA) is also commonly referred to as the spacer. When choosing the target binding region, we considered mainly 2 factors, namely the location of the PAM sequence and the suppression effect upon binding.
The research[1] shows CRISPRi suppression effect is the strongest 35nt upstream start codon of the coding region. However, the area upstream of our coding region is a generic constitutive promoter. To avoid non-specific binding, we compromised the suppression efficiency and chose a region shortly after the start codon, where suppression is only a few percents weaker than the ideal region. We found a PAM sequence (TGG) 27nt into gfp part BBa_E0040, which lead to a sgRNA binding region spanning 20bp, 7nt into CDS. To accommodate the PAM sequence in BBa_E1010 mrfp, the spacer is arranged on the opposite DNA strand, 14nt into the gene. When this part, specific to gfp is transcripted, GFP is suppressed.
Handle - tracrRNA
tracrRNA is an RNA loop that acts as a handle for dCas9 to hold onto. So that the dCas9 protein is delivered to the target site together with the sgRNA. Experiments have proved that tracrRNA is strictly required for Cas9-mediated DNA interference both in vitro and in vivo[4]. The tracrRNA forms a loop on the sgRNA after transcription to provide a scaffolding site for the dCas9 to form a duplex with the spacer.
Loop - artificial linker
Destroying the secondary structure of the handle in sgRNA could theoretically cause dissociation of the dCas9 protein from the sgRNA, thus, removing the suppression effect. The study mentioned above had proved this hypothesis correct. The team[1] then tried to design an artificial linker, which also forms a loop as a secondary structure, after the handle. After several trials and modifications, the research team discovered that extending the artificial loop, i.e. destroying the secondary structure, could further increase the derepression.
A corresponding antisense RNA (asRNA), K3017003, is responsible for reversing the repression effect on gfp induced by this sgRNA. Part of the antisense is complementary with the artificial linker of this sgRNA.
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| before modification | after modification |
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Secondary structure of sgRNA for gfp DNA binding |
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Characterizations
This part involve interaction with part BBa_K3017003 so they were being characterized as a pair. Using sgRNA-terminator BBa_K3017061 and asRNA-terminator-BBa_K3017060 as a model, we have characterized this part. Results show that the custom-designed sgRNA and asRNA pair hybridize as expected. We expect even higher hybridization rate. For details, please check out our part registry or experiment page
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.